Quantitative dna-based imaging and super-resolution imaging

ABSTRACT

The present disclosure provides, inter alia, methods and compositions (e.g., conjugates) for imaging, at high spatial resolution, targets of interest.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/934,759, filed Feb. 1, 2014, U.S.provisional application No. 61/884,126, filed Sep. 29, 2013, and U.S.provisional application No. 61/859,891, filed Jul. 30, 2013, each ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to the field of detection andquantification of targets.

BACKGROUND OF THE INVENTION

Far-field fluorescence microscopy has seen major advances since theadvent of methods that circumvent the classical diffraction limit, e.g.,super-resolution microscopy (refs. 1, 2). Most implementations switchmolecules between fluorescent ON- and OFF-states to allow consecutivelocalization of individual molecules. Switching is traditionallyobtained in one of two ways: “targeted” switching actively confines thefluorescence excitation to an area smaller than the diffraction of light(e.g., stimulated emission depletion microscopy, or STED (ref. 3)),whereas “stochastic” switching uses photoswitchable proteins(photoactivated localization microscopy, or PALM (ref. 4)) orphotoswitchable organic dyes (stochastic optical reconstructionmicroscopy, or STORM (ref. 1)). Although these methods offer enhancedspatial resolution, they tend to require either expensiveinstrumentation or highly specialized experimental conditions, and thushave not yet been developed into common biological laboratorytechniques.

SUMMARY OF THE INVENTION

The present disclosure provides, inter alia, methods, compositions(e.g., conjugates) and kits for imaging, at high or low spatialresolution, targets (e.g., biomolecules) of interest in, for example, acellular environment. The methods, compositions and kits of the presentdisclosure take advantage of repetitive, transient binding of short,labeled (e.g., fluorescently labeled) oligonucleotides (e.g., DNAoligonucleotides), or “imager” strands, to complementary “docking”strands, which are attached to targets of interest, in some embodiments,through an intermediate molecule such as an antibody such as a primaryor a secondary antibody, to obtain stochastic switching betweenfluorescent ON- and OFF-states (FIGS. 1A and 1B). In the unbound state,only background fluorescence from partially quenched (ref. 8) imagerstrands is observed (depicted by dimmer fluorescence of unbound imagerstrands in FIG. 1A). This is considered an “OFF” state. Upon binding andimmobilization of an imager strand, fluorescence emission is detectedusing, for example, total internal reflection (TIR) or highly inclinedand laminated optical sheet (HILO) microscopy (ref. 9). This isconsidered an “ON” state. In general, the methods, compositions and kitsas provided herein increase the imaging resolution and thus thesensitivity of detection. In some aspects, they also increase thespecificity as well as the number of utilizable fluorophores availablefor detecting targets of interest including but not limited to, e.g.,naturally-occurring biomolecules.

By linking a short docking strand to a binding partner (e.g., aprotein-binding moiety or a nucleic acid-binding moiety, whether primaryor secondary), such as an antibody including a primary and a secondaryantibody, different species of targets (e.g., biomolecules, optionallyin a cellular environment) can be labeled and subsequently detected byintroducing fluorescently-labeled imager strands that are complementaryto and bind to the docking strands through transient Watson-Crickinteractions. Unlike existing detection methods, the methods of thepresent disclosure are not limited by the number of spectrally distinctfluorophores available for detecting distinct targets (e.g.,biomolecules). Rather, the programmability of nucleic acid (e.g., DNAand/or RNA) molecules and sequential time-lapsed imaging are used hereinto provide images of up to hundreds of distinct species of targetsusing, in some embodiments, only a single optimized fluorophore.Further, these different species of targets (e.g., biomolecules) can bequantified using predictable kinetics of binding of singlefluorescently-labeled imager strands to their complementary targetdocking strands.

In some instances, the methods can be used to generate super-resolutionimages, significantly even without the need for a super-resolutionmicroscope. It should be understood that while the methods, compositionsand kits as provided herein may be described for use in super-resolutionimaging, they may also be used, in some embodiments, for imaging thatdoes not require super-resolution. Thus, in some embodiments, themethods, compositions and kits of the present disclosure may be used forimaging, generally.

In some aspects, provided herein is a protein-nucleic acid conjugate,comprising a protein linked to a docking strand that is capable oftransiently binding to a complementary labeled imager strand. In someaspects, provided herein is a protein-nucleic acid conjugate, comprisinga protein linked to a docking strand that is transiently bound to acomplementary labeled imager strand. Imager strands, in someembodiments, are labeled with a detectable label. The detectable labelmay be, for example, a fluorescent label or other detectable label,e.g., gold nanoparticle. While various aspects and embodiments hereinrefer to fluorescently-labeled imager strands, it should be understoodthat such fluorescent labels, in many instances, can be interchangedwith other detectable labels. Thus, in some embodiments, afluorescently-labeled imager strand (which may be detected by, forexample, fluorescent microscopy) may be interchanged with an imagerstrand labeled with, for example, gold nanoparticles (which may bedetected by, for example, dark field microscopy). It is also to beunderstood that the docking strands may be capable of transientlybinding a plurality of complementary labeled strands (e.g., the dockingstrand may comprise a plurality of binding sites for complementarylabeled strands).

In some embodiments, a method may be carried out involving a pluralityof docking strand and imager strand pairs. Such a method can be used todetect a plurality of targets (e.g., with each docking strand-imagerstrand pair corresponding to one target). The docking strand-imagerstrand pairs in the plurality must share an approximately equalprobability of hybridizing under a single environment or condition (asdefined for example by temperature, salt concentration, strand molarity,etc.), such that if there is an observed difference between the level ofbinding (and thus the detection) of a population of imager strands, anend user can conclude that such difference is a function of the amountof docking strand and thus ultimately the amount of target. In someembodiments, the docking and imager strands are typically selected suchthat their bound states have a thermal stability in the range of about+/−0.5 kcal/mol. With this range of thermal stability, it is possible toselect at least 200 orthogonal (e.g., different) sequences to be used inthese multiplexing methods. In some embodiments, a protein is anantibody such as a primary antibody or a secondary antibody, anantigen-binding antibody fragment, or a peptide aptamer.

In some embodiments, a protein is linked to the docking strand throughan intermediate linker. In some embodiments, the intermediate linkercomprises biotin and streptavidin.

In some embodiments, an antibody is a monoclonal antibody.

In some embodiments, a complementary fluorescently-labeled imager strandcomprises at least one fluorophore.

In some embodiments, a complementary labeled, optionally fluorescentlylabeled, imager strand is about 4 to about 30 nucleotides, or about 8 toabout 10 nucleotides, in length. In some embodiments, a complementarylabeled imager strand is longer than 30 nucleotides.

In this and other aspects and embodiments described herein, the dockingstrand may comprise a plurality of domains, each complementary to alabeled imager strand. The domains may be identical in sequence (andthus will bind to the identical imager strands) or they may be ofdifferent sequence (and thus may bind to imager strands that are notidentically labeled). Such domains may also be referred to herein asbinding sites for imager strands.

In some embodiments, a docking strand includes at least two or at leastthree domains, each respectively complementary to a labeled imagerstrand.

In some aspects, provided herein is a target bound to at least oneprotein-nucleic acid conjugate.

In some embodiments, the target is a protein. In some embodiments, thetarget is a nucleic acid (e.g., DNA or RNA).

In some aspects, provided herein is a plurality of protein-nucleic acidconjugates. In some embodiments, the plurality comprises at least twosubsets of the protein-nucleic acid conjugates, and the protein-nucleicacid conjugates of each subset bind to different targets.

In some aspects, provided herein is a composition or kit comprising aplurality of protein-nucleic acid conjugates, optionally wherein atleast one of the protein-nucleic acid conjugates is bound to at leastone target.

In some aspects, provided herein is a composition or kit comprising atleast one protein-nucleic acid conjugate that comprises a protein linkedto a docking strand, optionally wherein the at least one protein-nucleicacid conjugate is bound to a target, and at least one complementarylabeled, optionally fluorescently labeled, imager strand that istransiently bound to (or is capable of transiently binding to) the atleast one protein-nucleic acid conjugate.

In some embodiments, a composition or kit comprises at least twocomplementary labeled, optionally fluorescently labeled, imager strands,wherein the at least two complementary labeled imager strands areidentical. In some embodiments, the composition or kit comprises atleast two complementary labeled imager strands, wherein the at least twocomplementary labeled imager strands are different.

In some embodiments, the number of complementary labeled, optionallyfluorescently labeled, imager strands is less than, greater than orequal to the number of protein-nucleic acid conjugates.

In some embodiments, a composition or kit comprises at least 2, 3, 4, 5,6, 7, 9 or 10 different complementary labeled, optionally fluorescentlylabeled, imager strands. In some embodiments, the composition or kitcomprises at least 50 or at least 100 different complementaryfluorescently-labeled imager strands.

In some aspects, provided herein is a composition or a kit comprising a(e.g., one or more) docking strand and an (e.g., one or more) imagerstrand. The docking strand may be modified to include an affinity label,thereby facilitating its subsequent attachment to one or more bindingpartners, such as an antibodies. For example, the docking strand may bebiotinylated or it may be attached to avidin or streptavidin. Otheraffinity labels can be used instead. The imager strands may be labeled,such as fluorescently labeled. The imager strands may be a plurality ofidentical imager strands (e.g., with respect to sequence and label) orthey may be a plurality of different imager strands (e.g., with respectto sequence and label). The composition or kit may further comprise atarget-specific binding partner, such as an antibody. It is to beunderstood that the components may be bound to each other or they may beunbound, including physically separated from each other, in suchcompositions and kits. These and other compositions and kits may furthercomprise one or more buffers including oxygen scavengers.

In some aspects, provided herein is a composition or kit comprising anantibody-nucleic acid conjugate, wherein the antibody is a “secondaryantibody” having specificity for an antibody, typically specificity fora particular isotype or an Fc domain of an antibody from a particularspecies (e.g., a mouse antibody that is specific for a human IgG1antibody). The nucleic acid in the conjugate is a docking strand, asdescribed herein. The composition or kit may further comprise one ormore imager strands (or one or more subsets or populations of imagerstrands), as described herein. These and other compositions and kits mayfurther comprise one or more buffers including oxygen scavengers.

In some aspects, the present disclosure provides an antibody-DNAconjugate, comprising a monoclonal antibody linked to a docking strandthat is bound to a complementary labeled, optionally fluorescentlylabeled, imager strand, wherein the antibody and the docking strand areeach biotinylated and linked to each other through an avidin orstreptavidin linker or a biotin-streptavidin linker.

In some aspects, provided herein is an aptamer-nucleic acid conjugate,comprising a nucleic acid aptamer linked to a docking strand that istransiently bound to a complementary labeled, optionally fluorescentlylabeled, imager strand.

In some aspects, provided herein is a method of detecting a target in asample, the method comprising contacting a sample with (a) at least oneprotein-nucleic acid conjugate that comprises a protein linked to adocking strand and (b) at least one fluorescently-labeled imager strandthat is complementary to and transiently binds to the docking strand ofthe at least one protein-nucleic acid conjugate, and determining whetherthe at least one protein-nucleic acid conjugate binds to the target inthe sample. In some embodiments, the determining step comprises imagingtransient binding of the at least one fluorescently-labeled imagerstrand to the docking strand of the at least one protein-nucleic acidconjugate.

In some aspects, provided herein is a method of detecting a target in asample, the method comprising contacting a sample with (a) at least oneprotein-nucleic acid conjugate that comprises a protein linked to adocking strand and (b) at least one fluorescently-labeled imager strandthat is complementary to and transiently binds to the docking strand ofthe at least one protein-nucleic acid conjugate, and imaging transientbinding, optionally using time-lapsed imaging, of the at least onefluorescently-labeled imager strand to the docking strand of the atleast one protein-nucleic acid conjugate.

In some embodiments, a protein of the protein-nucleic acid conjugate isan antibody, an antigen-binding antibody fragment, or a peptide aptamer.In some embodiments, an antibody is a monoclonal antibody.

In some embodiments, a protein of the protein-nucleic acid conjugate islinked to the docking strand through an intermediate linker. In someembodiments, an intermediate linker comprises biotin and/orstreptavidin.

In some embodiments, a complementary fluorescently-labeled imager strandcomprises at least one fluorophore.

In some embodiments, a complementary labeled, optionally fluorescentlylabeled, imager strand is about 4 to about 10 nucleotides, or about 8 toabout 10 nucleotides in length.

In some embodiments, a sample is a cell or cell lysate.

In some embodiments, a target is a protein. In some embodiments, atarget is a nucleic acid (e.g., DNA or RNA).

In some embodiments, a target is obtained from a cell or cell lysate.

In some aspects, provided herein is a method of detecting at least oneor at least two targets in a sample, the method comprising contacting asample with (a) at least two protein-nucleic acid conjugates, eachcomprising a protein linked to a docking strand, and (b) at least twolabeled (optionally spectrally distinct, or fluorescently labeled, orspectrally distinct and fluorescently labeled) imager strands that arecomplementary to and transiently bind to respective docking strands ofthe at least one, or at least, two different protein-nucleic acidconjugates and determining whether the at least two protein-nucleic acidconjugates bind to at least two targets in the sample. In someembodiments, the determining step comprises, in the following order,imaging transient binding of one of the at least two labeled imagerstrands to a docking strand of one of the at least two protein-nucleicacid conjugates to produce a first image (e.g., of a fluorescentsignal), and imaging transient binding of another of the at least twolabeled imager strands to a docking strand of another of the at leasttwo protein-nucleic acid conjugates to produce at least one other image(e.g., of a fluorescent signal). In some embodiments, the method furthercomprises combining the first image and the at least one other image toproduce a composite image of signal (e.g., fluorescent signal), whereinthe signal of the composite image is representative of the at least twotargets.

In some embodiments, a protein of the protein-nucleic acid conjugate isan antibody, an antigen-binding antibody fragment, or a peptide aptamer.In some embodiments, an antibody is a monoclonal antibody.

In some embodiments, a protein of the protein-nucleic acid conjugate islinked to the docking strand through an intermediate linker. In someembodiments, an intermediate linker comprises biotin and streptavidin.

In some embodiments, each of the at least two spectrally distinct,fluorescently-labeled imager strands comprises at least one fluorophore.

In some embodiments, each of the at least two labeled, optionallyspectrally distinct, fluorescently labeled, imager strands is about 4 toabout 10 nucleotides, or about 8 to about 10 nucleotides in length.

In some embodiments, a sample is a cell or cell lysate.

In some embodiments, at least two targets are proteins. In someembodiments, at least two targets are nucleic acids (e.g., DNA or RNA).

In some embodiments, at least two targets are obtained from a cell orcell lysate.

In some aspects, provided herein is a method of detecting at least one,or at least two, protein targets in a sample, comprising (a) contactinga sample with at least two protein-nucleic acid conjugates, eachcomprising a protein linked to a docking strand and (b) sequentiallycontacting the sample with at least two labeled (e.g., optionallyspectrally indistinct, or fluorescently labeled, or spectrally distinctand fluorescently labeled) imager strands that are complementary to andtransiently bind to respective docking strands of the at least twoprotein-nucleic acid conjugates, and determining whether the at leastone or at least two protein-nucleic acid conjugates bind to at least twotargets in the sample. In some embodiments, a method comprises, in thefollowing ordered steps, contacting the sample with a firstprotein-nucleic acid conjugate and at least one other protein-nucleicacid conjugate, contacting the sample with a first labeled, optionallyfluorescently labeled, imager strand that is complementary to andtransiently binds to the docking strand of the first protein-nucleicacid conjugate, imaging the sample to obtain a first image, optionallyusing time-lapsed imaging, removing the first labeled imager strand,contacting the sample with at least one other labeled imager strand thatis complementary to and transiently binds to the docking strand of theat least one other protein-nucleic acid conjugate, and imaging thesample to obtain at least one other image, optionally using time-lapsedimaging.

In some embodiments, a method comprises, in the following ordered steps,contacting a sample with a first protein-nucleic acid conjugate,contacting the sample with a first labeled, optionally fluorescentlylabeled, imager strand that is complementary to and transiently binds tothe docking strand of the first protein-nucleic acid conjugate, imagingthe sample to obtain a first image, optionally using time-lapsedimaging, removing the first labeled imager strand, contacting the samplewith at least one other protein-nucleic acid conjugate, contacting thesample with at least one other labeled, optionally fluorescentlylabeled, imager strand that is complementary to and transiently binds tothe docking strand of the at least one other protein-nucleic acidconjugate, and imaging the sample to obtain at least one other image,optionally using time-lapsed imaging.

In some embodiments, a method further comprises determining whether thefirst protein-DNA conjugate binds to a first target and/or whether theat least one other protein-DNA conjugate binds to at least one othertarget.

In some embodiments, a method further comprises assigning a pseudo-colorto the signal (e.g., fluorescent signal) in a first image, and assigningat least one other pseudo-color to the fluorescent signal in the atleast one other image.

In some embodiments, a method further comprises combining a first imageand the at least one other image to produce a composite image of thepseudo-colored signals, wherein the pseudo-colored signals of thecomposite image are representative of the at least two targets.

In some embodiments, the protein of the protein-nucleic acidconjugate(s) is an antibody, an antigen-binding antibody fragment, or apeptide aptamer. In some embodiments, the antibody is a monoclonalantibody.

In some embodiments, the protein of the protein-nucleic acidconjugate(s) is linked to the docking strand through an intermediatelinker. In some embodiments, the intermediate linker comprises biotinand/or streptavidin.

In some embodiments, each of the fluorescently-labeled imager strandscomprises at least one fluorophore.

In some embodiments, each of the fluorescently-labeled imager strands isabout 4 to about 30 nucleotides, or about 8 to about 10 nucleotides inlength.

In some embodiments, a sample is a cell or cell lysate.

In some embodiments, a target(s) is a protein. In some embodiments, atarget(s) is a nucleic acid (e.g., DNA or RNA).

In some embodiments, a target(s) is obtained from a cell or cell lysate.

In some aspects, provided herein is a method of detecting a target,optionally a naturally-occurring biomolecule, comprising contacting asample containing at least one target, optionally a naturally-occurringbiomolecule, with (a) at least one BP-NA conjugate, optionally eachBP-NA conjugate comprising a protein or nucleic acid linked to a dockingstrand, and (b) at least one labeled, optionally fluorescently labeled,imager strand that is complementary to and transiently binds the dockingstrand of the at least one BP-NA conjugate, and determining whether theat least one BP-NA conjugate binds to at least one target, optionally anaturally-occurring biomolecule, in the sample. In this and otheraspects or embodiments described herein, it is to be understood that themethod may be carried out using a sample that is suspected of containingat least one target or a sample that an end-user desires to analyze forthe presence of the at least one target without any prior knowledge ofthe sample respecting its likelihood of containing the target.

In some embodiments, the determining step comprises imaging transientbinding of the at least one labeled, optionally fluorescently labeled,imager strand to the docking strand of the at least one BP-NA conjugate.

In some embodiments, a sample is a cell or cell lysate.

In some embodiments, an at least one target, optionally anaturally-occurring biomolecule, is obtained from a cell or cell lysate.

In some embodiments, a protein is an antibody, an antigen-bindingantibody fragment, or a peptide aptamer. In some embodiments, anantibody is a monoclonal antibody.

In some embodiments, a protein is linked to the docking strand throughan intermediate linker. In some embodiments, an intermediate linkercomprises biotin and/or streptavidin.

In some embodiments, a nucleic acid is a nucleic acid aptamer.

In some embodiments, a fluorescently-labeled imager strand comprises atleast one fluorophore.

In some embodiments, an imager strand, optionally afluorescently-labeled imager strand, is about 4 to about 30, or about 8to about 10 nucleotides in length.

In some aspects, provided herein is a method of detecting a target,optionally a naturally-occurring biomolecule, comprising contacting asample containing at least two targets, optionally naturally-occurringbiomolecules, with (a) at least two different BP-NA conjugates,optionally each BP-NA conjugate comprising a protein or nucleic acidlinked to a DNA docking strand, and (b) at least two labeled (optionallyspectrally indistinct, or fluorescently labeled, or spectrally distinctand fluorescently labeled) imager strands that are complementary to andtransiently bind to respective docking strands of the at least two BP-NAconjugates, and determining whether the at least two BP-NA conjugatesbind to at least one or at least two naturally-occurring biomolecules inthe sample.

In some embodiments, a method comprises, in the following ordered steps,contacting the sample with a first BP-NA conjugate and at least oneother BP-NA conjugate, contacting the sample with a first labeled,optionally fluorescently labeled, imager strand that is complementary toand transiently binds to the docking strand of the first BP-NAconjugate, imaging the sample to obtain a first image, optionally usingtime-lapsed imaging, removing the first labeled imager strand,contacting the sample with at least one other labeled, optionallyfluorescently labeled, imager strand that is complementary to andtransiently binds to the docking strand of the at least one other BP-NAconjugate, and imaging the sample to obtain at least one other image,optionally using time-lapsed imaging.

In some embodiments, a method comprises, in the following ordered steps,contacting the sample with a first BP-NA conjugate, contacting thesample with a first labeled, optionally fluorescently labeled, imagerstrand that is complementary to and transiently binds to the dockingstrand of the first BP-NA conjugate, imaging the sample to obtain afirst image, optionally using time-lapsed imaging, removing the firstlabeled imager strand, contacting the sample with at least one otherBP-NA conjugate, contacting the sample with at least one other labeled,optionally fluorescently labeled, imager strand that is complementary toand transiently binds to the docking strand of the at least one otherBP-NA conjugate, and imaging the sample to obtain a at least one otherimage, optionally using time-lapsed imaging.

In some embodiments, a method further comprises determining whether thefirst protein DNA conjugate binds to a first target, optionally anaturally-occurring biomolecule, and/or whether the at least one otherprotein-DNA conjugate binds to at least one other target, optionally anaturally-occurring biomolecule.

In some embodiments, a method further comprises assigning a pseudo-colorto the signal (e.g., fluorescent signal) in a first image, and assigningat least one other pseudo-color to the signal (e.g., fluorescent signal)in at least one other image.

In some embodiments, a method further comprises combining a first imageand at least one other image to produce a composite image ofpseudo-colored signals, wherein the pseudo-colored signals of thecomposite image are representative of at least one, or at least two,targets (e.g., naturally-occurring biomolecules).

In some aspects, provided herein is a method of determining the numberof targets in a test sample, comprising obtaining a sample thatcomprises targets transiently bound directly or indirectly to labeled,optionally fluorescently labeled, imager strands, obtaining atime-lapsed image, optionally a time-lapsed diffraction-limitedfluorescence image, of the sample, performing spot detection (e.g.,fluorescence spot detection) and localization (e.g., through the use ofGaussian fitting) on the diffraction-limited image to obtain ahigh-resolution image of the sample, calibrating k_(on)·c_(imager),optionally using a control sample with a known number of targets,wherein k_(on) is a second order association constant, and c_(imager) isconcentration of labeled (e.g., fluorescently labeled) imager strands inthe test sample, determining variable τ_(d), optionally by fitting thefluorescence OFF-time distribution to a cumulative distributionfunction, and determining the number of test targets in the sample basedon the equation, number of test targets=(k_(on)·c_(imager)·τ_(d))⁻¹.

In some aspects, provided herein is a method of determining a relativeamount of targets in a test sample, comprising obtaining a sample thatcomprises targets transiently bound directly or indirectly to labeledimager strands, obtaining a time-lapsed image of the sample, performingspot detection and localization on the image to obtain a high-resolutionimage of the sample, determining variable τ_(d), and determining therelative amount of two or more test targets in the sample based onτ_(d).

In some embodiments, test targets are protein targets.

In some embodiments, protein targets are bound to protein-nucleic acidconjugates that comprise a protein linked to a docking strand, and thelabeled (e.g., fluorescently labeled) imager strands are complementaryto and transiently bind to respective docking strands of theprotein-nucleic acid conjugates.

In some embodiments, the protein of the protein-nucleic acid conjugateis an antibody, an antigen-binding antibody fragment, or a peptideaptamer.

In some embodiments, test targets are single-stranded nucleic acids.

In some embodiments, single-stranded nucleic acids are DNA or RNA.

In some embodiments, each of the fluorescently-labeled imager strandscomprises at least one fluorophore.

In some embodiments, each of the labeled, optionally fluorescentlylabeled, imager strands is about 4 to about 30 nucleotides, or about 8to about 10 nucleotides in length.

In some embodiments, a time-lapsed fluorescence image is obtained over aperiod of about 25 minutes.

In some embodiments, the number of test targets is determined with anaccuracy of greater than 90%.

In some aspects, provided herein is a single-stranded DNA probecomprising a target binding domain of about 20 nucleotides in lengthlinked, optionally at its 3′ end, to a docking domain of at least one,at least two, or at least three subdomains, wherein the at at least one,least two, or at least three subdomains are respectively complementaryto at least one, at least two, or at least three labeled, optionallyfluorescently labeled, imager strands of about 4 to about 30, or about 8to 10 nucleotides in length, and wherein the target binding domain isbound to a complementary domain of a single-stranded mRNA target strand.

In some embodiments, at least one of the at least one, at least two, orat least three subdomains is transiently bound to at least one labeled,optionally fluorescently labeled, imager strand.

In some aspects, provided herein is a method of performing driftcorrection for a plurality of images, wherein each of the plurality ofimages comprises a frame of a time sequence of images, wherein the timesequence of images captures a plurality of transient events, the methodcomprising determining a time trace for each of a plurality of driftmarkers identified in the plurality of images, wherein a time trace foreach drift marker corresponds to movement of an object in the image overthe time sequence of images, determining, with at least one computerprocessor, a first drift correction from at least one of the pluralityof drift markers based, at least in part, on the time traces for theplurality of drift markers, determining a time trace for each of aplurality of geometrically-addressable marker sites from a plurality ofdrift templates identified from the plurality of images, wherein eachdrift template in the plurality of drift templates describes ageometrical relationship between the plurality ofgeometrically-addressable marker sites of transient events in the drifttemplate, determining a second drift correction based, at least in part,on the time traces for the plurality of geometrically-addressable markersites from the plurality of drift templates, correcting the plurality ofimages based, at least in part, on the first drift correction and thesecond drift correction, and outputting a final image based on thecorrected plurality of images.

In some embodiments, a method further comprises identifying a pluralityof localizations in each of the plurality of images, creating atwo-dimensional histogram of the plurality of localizations, andidentifying locations of the plurality of drift markers based, at leastin part, on the two-dimensional histogram, wherein determining the timetraces for each of the plurality of drift markers comprises determiningthe time traces based, at least in part, on the locations of theplurality of drift markers.

In some embodiments, identifying a plurality of localizations comprisesidentifying a plurality of spots on each of the plurality of images, anddetermining a fitted center position of each of the plurality of spotsusing a local Gaussian fitting algorithm, wherein each of the pluralityof localizations comprises the spot identified on an image and itsassociated fitted center position.

In some embodiments, each of the plurality of localizations furthercomprises a detected photon count corresponding to the localization.

In some embodiments, creating the two-dimensional histogram of theplurality of localizations comprises binning all localizations into atwo-dimensional grid and using a total number of localizations in eachbin as a histogram count.

In some embodiments, creating the two-dimensional histogram of theplurality of localizations comprises binning all localizations into atwo-dimensional grid and using a total number of photon count of theplurality of localizations in each bin as a histogram count.

In some embodiments, identifying locations of the plurality of driftmarkers based, at least in part, on the two-dimensional histogramcomprises at least one of the following: binarizing the two-dimensionalhistogram using one or more selection criteria, wherein the one or moreselection criteria include a lower-bound threshold of a histogram valueor a upper-bound threshold of a histogram value; partitioning thebinarized image into partitions and filtering the partitions based onone or more selection criteria, wherein the one or more selectioncriteria include one or more of a lower-bound threshold of an area of apartition area, an upper-bound threshold of the area, a lower-bound oran upper-bound of a longest or shortest linear dimension of a partitionlongest, and a lower-bound or an upper-bound of an eccentricity of apartition; and expanding and shrinking the binarized image using one ormore binary image operations, wherein the one or more binary imageoperations include one or more of the following: dilate, erode, bridge,close, open, fill, clean, top-hat, bottom-hat, thicken, thin, and more.

In some embodiments, determining a first drift correction based, atleast in part, on the time traces for the plurality of drift markerscomprises: determining a relative time trace for each of the pluralityof drift markers, wherein the relative time trace is determined bycomparing the time trace for the drift marker with the average positionof the same trace; and determining a combined time trace based on therelative time traces for each of the plurality of drift markers, whereindetermining the first drift correction based, at least in part, on thetime traces for the plurality of drift markers comprises determining thefirst drift correction based, at least in part, on the relative timetraces for each of the plurality of drift markers.

In some embodiments, determining the first drift correction based, atleast in part, on the relative time traces for each of the plurality ofdrift markers comprises performing a weighted average of the relativetime traces for each of the plurality of drift markers.

In some embodiments, performing the weighted average comprises:determining a quality score for each of the relative time traces,wherein the quality score is determined based, at least in part, on ameasure of variability over time associated with the time trace and/or ameasure of localization uncertainty of individual localizations withinthe time trace.

In some embodiments, the measure of variability over time comprises astandard deviation of the time trace over time.

In some embodiments, the measure of localization uncertainty ofindividual localizations comprises, at least in part, an estimate ofuncertainty from a Gaussian fitting or a comparison with othersimultaneous localizations, wherein the other simultaneous localizationsare from within a same image and from other time traces from theplurality of drift markers, wherein the comparison comprises a mean andstandard deviation of all simultaneous localizations.

In some embodiments, the method further comprises determining that afirst drift marker of the plurality of drift markers is not present inat least one frame of the time sequence of images, and linearlyinterpolating the time trace for the first drift marker for the at leastone frame to produce a smoothed time trace for the first drift marker.

In some embodiments, determining a time trace for each of a plurality ofgeometrically-addressable marker sites from a plurality of drifttemplates identified from the plurality of images comprises: identifyinga plurality of localizations in each of the plurality of images;creating a two-dimensional histogram of the plurality of localizations;and identifying the plurality of drift templates based, at least inpart, on the two-dimensional histogram, wherein identifying theplurality of drift templates comprises evaluating the two-dimensionalhistogram using an lower-bound and/or an upper-bound threshold in ahistogram count.

In some embodiments, determining a time trace for each of a plurality ofgeometrically-addressable marker sites from a plurality of drifttemplates identified from the plurality of images comprises determininga time trace for each of a plurality of geometrically-addressable markersites within each of the plurality of drift templates, and whereindetermining the second drift correction comprises determining the seconddrift correction based, at least in part, on the time traces for each ofthe plurality of marker sites within each of the plurality of drifttemplates.

In some embodiments, determining the second drift correction based, atleast in part, on the time traces for each of the plurality ofgeometrically-addressable marker sites from each of the plurality ofdrift templates comprises: identifying a plurality ofgeometrically-addressable marker sites within each of the plurality ofdrift templates; and determining a relative time trace for each of aplurality of geometrically-addressable drift markers for each of theplurality of drift templates, wherein determining the second driftcorrection based, at least in part, on the time traces for the pluralityof drift templates comprises determining the second drift correctionbased, at least in part, on the relative time traces for each of theplurality of drift markers within each of the plurality of drifttemplates.

In some embodiments, identifying a plurality of geometricallyaddressable marker sites from each of the plurality of drift templatescomprises determining a plurality of marker sites based on, at least inpart, a two-dimensional histogram of the plurality of localizations inthe corresponding drift template, and/or one or more selection criteria,wherein the one or more selection criteria include one or more of atotal number of localizations, a surface density of localizations, andstandard deviation of localizations.

In some embodiments, determining the second drift correction based, atleast in part, on the relative time traces for each of the plurality ofdrift markers within each of the plurality of drift templates comprisesperforming a weighted average of the relative time traces for each ofthe plurality of drift markers within each of the drift templates.

In some embodiments, performing the weighted average comprises:

determining a quality score for each of the relative time traces,wherein the quality score is determined based, at least in part, on ameasure of variability over time associated with the time trace and/or alocalization uncertainty within the time trace.

In some embodiments, the measure of variability over time comprises astandard deviation of the time trace over time.

In some embodiments, the measure of localization uncertainty ofindividual localizations comprises an estimate of uncertainty from aGaussian fitting or a comparison with other simultaneous localizations,wherein the other simultaneous localizations are from within a sameimage and from the other time traces from the plurality of marker sitesfrom the plurality of drift templates, wherein the comparison comprisesa mean and standard deviation of all simultaneous localizations.

In some embodiments, correcting the plurality of images based, at leastin part, on the first drift correction and the second drift correctioncomprises correcting the plurality images using the first driftcorrection to produce a first corrected plurality of images, and whereindetermining a time trace for each of a plurality of drift templatesidentified from the plurality of images comprises determining a timetrace for each of the plurality of drift templates identified from thefirst corrected plurality of images.

In some embodiments, a method further comprises smoothing the firstdrift correction prior to correcting the plurality of images using thefirst drift correction.

In some embodiments, smoothing the first drift correction comprisesprocessing the first drift correction using local regression with awindow determined by a characteristic drift time scale of the firstdrift correction.

In some embodiments, a method further comprises smoothing the seconddrift correction prior to correcting the plurality of images using thesecond drift correction.

In some embodiments, smoothing the second drift correction comprisesprocessing the second drift correction using local regression with awindow determined by a characteristic drift time scale of the seconddrift correction.

In some embodiments, a method further comprises selecting a single driftmarker of the plurality of drift markers; and determining a third driftcorrection based, at least in part, on the selected single drift marker,wherein correcting the plurality of images comprises correcting theplurality of images based, at least in part, on the third driftcorrection.

In some embodiments, correcting the plurality of images based, at leastin part, on the third drift correction is performed prior to correctingthe plurality of images based, at least in part on the first driftcorrection and the second drift correction.

In some embodiments, a method further comprises identifying locations ofa first plurality of points in a first image of the plurality of frames;identifying locations of a second plurality of points in a second imageof the plurality of images, wherein the second image corresponds to aneighboring frame of the first image in the time sequence of images; anddetermining a fourth drift correction based, at least in part, ondifferences between the locations of the first plurality of points andthe second plurality of points, wherein correcting the plurality ofimages comprises correcting the plurality of images based, at least inpart, on the fourth drift correction.

In some embodiments, the second image corresponds to a frame immediatelyfollowing the frame corresponding to the first image in the timesequence of images.

In some embodiments, determining the fourth drift correction based, atleast in part, on differences between the locations of the firstplurality of points and the second plurality of points comprises:creating a histogram of distances between the locations of the firstplurality of points and the second plurality of points; determiningbased, at least in part, on the histogram, pairs of points between thefirst image and the second image that correspond to the same transientevent; and determining a location offset between each of the determinedpairs of points, wherein determining the fourth drift correction isbased on a vector average of the location offsets for each of thedetermined pairs of points.

In some embodiments, the plurality of images correspond to DNA-basedimages and wherein the plurality of transient events are binding eventsbetween an imaging strand and a DNA docking strand.

In some embodiments, the imaging strand is a fluorescent imaging probeconfigured to fluoresce when associated with the DNA docking strand.

In some embodiments, at least one of the drift markers is a DNA basednanostructure.

In some embodiments, the DNA based nanostructure is a DNA origaminanostructure with docking strands.

In some embodiments, at least one of the drift templates is a DNA basednanostructure.

In some embodiments, the DNA based nanostructure is a DNA origaminanostructure with docking strands.

In some embodiments, at least one of the drift templates is athree-dimensional drift template.

In some embodiments, the three-dimensional drift template is atetrahedron.

In some embodiments, at least one of the drift templates includesmultiple colors corresponding to different types of transient events.

In some embodiments, the different types of transient events include afirst binding event of a first imaging strand with a first type of DNAdocking strand and a second binding event of a second imaging strandwith a second type of DNA docking strand.

In some embodiments, outputting the final image comprises displaying thefinal image on a display.

In some embodiments, outputting the final image comprises sending thefinal image to a computer via at least one network.

In some embodiments, outputting the final image comprises storing thefinal image on at least one storage device.

In some aspects, provided herein is a non-transitory computer readablemedium encoded with a plurality of instructions that, when executed byat least one computer processor, performs a method of performing driftcorrection for a plurality of images, wherein each of the plurality ofimages comprises a frame of a time sequence of images, wherein the timesequence of images captures a plurality of transient events, the methodcomprising: determining a time trace for each of a plurality of driftmarkers identified in the plurality of images, wherein a time trace foreach drift marker corresponds to movement of an object in the image overthe time sequence of images; determining a first drift correction fromat least one of the plurality of drift markers based, at least in part,on the time traces for the plurality of drift markers; determining atime trace for each of a plurality of geometrically-addressable markersites from a plurality of drift templates identified from the pluralityof images, wherein each drift template in the plurality of drifttemplates describes a geometrical relationship between the plurality ofgeometrically-addressable marker sites of transient events in the drifttemplate; determining a second drift correction based, at least in part,on the time traces for the plurality of geometrically-addressable markersites from the plurality of drift templates; correcting the plurality ofimages based, at least in part, on the first drift correction and thesecond drift correction; and outputting a final image based on thecorrected plurality of images.

In some aspects, provided herein is a computer, comprising: an inputinterface configured to receive a plurality of images, wherein each ofthe plurality of images comprises a frame of a time sequence of images,wherein the time sequence of images captures a plurality of transientevents; at least one processor programmed to: determine a time trace foreach of a plurality of drift markers identified in the plurality ofimages, wherein a time trace for each drift marker corresponds tomovement of an object in the image over the time sequence of images;determine a first drift correction from at least one of the plurality ofdrift markers based, at least in part, on the time traces for theplurality of drift markers; determine a time trace for each of aplurality of geometrically-addressable marker sites from a plurality ofdrift templates identified from the plurality of images, wherein eachdrift template in the plurality of drift templates describes ageometrical relationship between the plurality ofgeometrically-addressable marker sites of transient events in the drifttemplate; determine a second drift correction based, at least in part,on the time traces for the plurality of geometrically-addressable markersites from the plurality of drift templates; correct the plurality ofimages based, at least in part, on the first drift correction and thesecond drift correction; and determine a final image based on thecorrected plurality of images; and output interface configured to outputthe final image.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows microtubule-like DNA origami polymers “labeled” withsingle-stranded DNA docking strands on a pair of opposite faces (coloredin dark gray) spaced approximately 16 nanometers (nm) apart.Complementary fluorescently-labeled imager strands transiently bind fromsolution to the docking strands. Biotinylated DNA strands (present onthe bottom two center helices) are used to bind the microtubule-like DNAstructures to glass surfaces for fluorescence imaging. FIG. 1B shows agraph demonstrating that transient binding of fluorescently-labeledimager strands to the docking strands produces fluorescence“blinking”(fluorescence intensity vs. time trace). This blinking is usedto consecutively localize points below the diffraction limit. FIG. 1Cshows a transmission electron microscope (TEM) image of DNA origamipolymers with a measured width of 16±1 nm (mean±stdv) [scale bar: 40nm]. FIG. 1D shows super-resolution fluorescence images obtained usingCy3b-labeled imager strands (15,000 frames, 5 Hz frame rate). Twodistinct lines are visible [scale bars: 40 nm]. FIG. 1E shows across-sectional histogram of highlighted areas <i> and <ii> in FIG. 1D(arrows denote histogram direction), which show that the designeddistance of approximately 16 nm is clearly resolved (full width at halfmaximum (FWHM) of each distribution is observed to be approximately 9nm).

FIG. 2 shows an example of a biomolecule labeling scheme of the presentdisclosure where a protein (e.g., protein target) is labeled withantibody-DNA conjugates of the present disclosure and complementaryfluorescently-labeled imager strands. The antibodies are linked to thedocking strands through a linker that contains biotin and streptavidin(e.g., biotin-streptavidin-biotin linker).

FIG. 3A shows a super-resolution image of a microtubule network inside afixed HeLa cell using an antibody-DNA conjugate and Atto655-labeledimager strands (10,000 frames, 10 Hz frame rate) [scale bar: 5 μm]. FIG.3B shows a high magnification image of the highlighted area in FIG. 3A[scale bar: 1 μm]. FIG. 3C shows a diffraction-limited representation ofthe same area in FIG. 3B. Arrows highlight positions where the increasein resolution of the image is clearly visible. Adjacent microtubuleswith an apparent width of approximately 46 nm at position <i> in FIG. 3Bare separated by approximately 79 nm [scale bar: 1 μm]. FIG. 3D shows adual-color super-resolution image (15,000 frames, 10 Hz frame rate) ofmicrotubules and mitochondria inside a fixed HeLa cell obtained usingantibody-DNA conjugates, Cy3b-labeled imager strands for microtubules(linear-like structures) and Atto655-labeled imager strands formitochondria (patch-like structures) [scale bar: 5 μm]. FIG. 3E shows ahigh magnification image of the highlighted area in FIG. 3D [scale bar:1 μm]. FIG. 3F shows a diffraction-limited image of the same area shownin FIG. 3E [scale bar: 1 μm].

FIG. 4A shows one embodiment of the present disclosure using spectrallyindistinct imager strands (e.g., each labeled with the same colorfluorophore). In step [1], three different species of docking strands(a,b,c) label a surface. Such labeling may occur using the dockingstrand alone or linked to a protein-binding (e.g., antibody) or anucleic acid-binding molecule that binds to the surface/biomolecule ofinterest. In step [2], multiple copies of the imager strand a* areintroduced (where a* has a sequence complementary to a), and pointslabeled with docking strands a are imaged. In step [3], copies of theimager strand a* are flushed away, and imager strand b* is introduced toimage the b labeled points. Images are obtained, and imager strands b*are washed away. In step [4], c labeled points are imaged in the samemanner. In step [5], images from [2-4] are assigned pseudo-colors andcombined to create the final image. Although pseudo-colors may be usedin the final rendering of the image, all imager strands are actuallylabeled with the same color dye (e.g., fluorophore). FIGS. 4B(1)-4B(3)show that reducing the image density simultaneously increases theachievable resolution by up to a factor of 2√{square root over (2)} ln2≈2.35. Here, the resolution, previously defined as the FWHM of thereconstructed localizations, can be understood as the standard deviationof the localization as in localization microscopy with sparse points.FIG. 4B(1) shows seven points in a linear geometry spaced at 10 nm(top). Simulated super-resolution data with approximately 14 nmresolution (center). The points cannot be resolved. Cross-sectionalhistogram data shows a broad peak (bottom). FIGS. 4B(2) and 4B(3) showthat imaging every other site allows the localization of individualspots. These localizations can then be combined to form the final imageof the full pattern. FIG. 4C shows an image of a DNA origami structurethat displays docking strands spaced at 10 nm intervals.

FIG. 5A shows one embodiment of the present disclosure using DNA origamistructures with different species of docking strands at designatedpositions resembling numbers 0-3 (0, 1, 2 and 3). For each round, therespective imager strand sequence is added to an imaging chamber, imageacquisition is carried out and the imager strands are washed out. Ineach imaging round the designed number is imaged, showing a verysequence-specific interaction with no crosstalk between different rounds[Scale bar: 50 nm]. Note that all imager strands are labeled with thesame color dye, though each structure (e.g., 0-3 (0, 1, 2 and 3)) isrendered a distinct color (e.g., purple, yellow, blue, or red; colorrendering not shown). FIGS. 5B(i)-(v) show another embodiment of thepresent disclosure using DNA origami structures with different speciesof docking strands at designated positions resembling numbers 0-9 (0, 1,2, 3, 4, 5, 6, 7, 8 and 9). FIG. 5B(i) shows an exchange-PAINT schematicshowing sequential imaging of multiple targets using imager strandslabeled with the same fluorophore. FIG. 5B(ii) shows a schematic of aDNA origami (70 100 nm) displaying docking strands that resemble digit4. FIG. 5B(iii) shows a combined overview image of all tenExchange-PAINT cycles, demonstrating specific interaction with therespective target with no crosstalk between imaging cycles. Scale bar:250 nm. FIG. 5B(iv) shows a four-“color” image of digits 0 to 3 that areall present on the same DNA origami (10,000 frames each, 5 Hz framerate; schematic at the bottom). Scale bar: 25 nm. FIG. 5B(v) showspseudocolor images of ten different origami structures, each rendered adistinct color (e.g., orange, green, blue, purple, pink, etc.; colorrendering not shown), displaying digits 0 to 9 in one sample with highresolution (FWHM of bar-like features <10 nm) and specificity. Imageobtained using only one fluorophore (Cy3b) through ten imaging-washingcycles (imaging: 7,500 frames per cycle, 5 Hz frame rate; washing: 1-2minutes per cycle). Scale bar: 25 nm.

FIG. 6A shows an experimental schematic for one embodiment of thepresent disclosure using fixed HeLa cells, where in one round, dockingstrands are bound to a target, labeled imager strands are then added, animage is acquired, and the imager strands are washed away. Each round isrepeated with docking strands specific for different targets along withdifferent labeled imager strands. The docking strands may be used aloneor linked to a protein-binding (e.g., antibody) or a nucleicacid-binding molecule that binds to the target of interest. FIG. 6Bshows two rounds of a method of the present disclosure usingCy3b-labeled imager strands in fixed HeLa cells. Here, microtubules(green pseudo-color; color rendering not shown) were labeled withdocking sequence a and mitochondria (magenta pseudo-color; colorrendering not shown) with orthogonal docking sequence b. FIG. 6C showstwo rounds of a method of the present disclosure using ATTO655-labeledimager strands performed in fixed HeLa cells similar to FIG. 6B. [Scalebars: 5 μm]. Note that all imager strands are labeled with the samecolor dye.

FIG. 7A shows that fluorescently-labeled imager strands transiently bindfrom solution to complementary docking strands on a structure ormolecule of interest. The transient binding produces an apparentblinking as shown in the binding vs. time trace with characteristicfluorescence ON- and OFF-times (τ_(b)and τ_(d), respectively). Thedetected binding frequency of imager strands from solution is linearlydependent on the number of available docking strands in a given imagearea (i.e., the more docking strands, the higher the binding frequency).The time in-between binding events, i.e., the fluorescence OFF-time(τ_(d)), is inversely proportional to the number of docking strands.FIG. 7B shows that the average fluorescence OFF-time (τ_(d)) can bedetermined by calculating a cumulative distribution function (CDF) forthe OFF-time distribution. Given a known association constant k_(on) andimager strand concentration c, the number of binding sites can becalculated by: number of binding sites=(τ_(d)·c·k_(on))⁻¹. FIG. 7C showsa super-resolution image of DNA origami structures designed to display13 binding sites as a proof-of-concept platform. The incorporationefficiency for docking sites is not 100% leading to a distribution ofactually incorporated sites (FIG. 7D(1)). The structures serve as anideal test system, as the number of displayed docking sites can bedetermined visually by counting the number of spots (direct counting)and comparing it with the corresponding number of sites calculated usingthe proposed binding kinetic analysis. FIG. 7D(1) shows the binding sitedistribution for 377 origami structures obtained by direct visualcounting. FIG. 7D(2) shows the binding site distribution for the samestructures obtained by binding kinetic analysis (kinetics) of thepresent disclosure. FIG. 7D(3) shows the “offset” between direct andkinetic counting: the counting “error” or uncertainty for the method ofthe present disclosure is less than 7% (determined by the coefficient ofvariation of the Gaussian distribution).

FIG. 8A shows mRNA molecules of interest in fixed Escherichia coli cellstagged using docking strands in a FISH-like hybridization scheme. FIG.8B shows a readout scheme used to determine the binding frequency foreach imaging color. The intensity vs. time profile of each single mRNAlocation yields a specific transient binding pattern (blinking) percolor. The frequency of binding events depends on the number of bindingsites allowing the use of the binding frequency to distinguish betweendifferent integer numbers of binding sites. FIG. 8C shows an in vitroproof-of-principle experiment on DNA origami structures displaying 3, 9,22 and 44 binding sites for each of the red, green, and blue imagerstrands, respectively (color rendering not shown). The different bindinglevels are clearly distinguishable for each color, suggesting 4 possible“frequency levels” per color, yielding up to 124 different possiblecombinations for barcoding, e.g., mRNA molecules inside cells. Thebarcoding space can be increased by using the fluorescence ON-time as anadditional coding entity.

FIG. 9 shows a graph demonstrating that the fluorescence ON-time(related to the dissociation rate k_(off)) can be tuned independently ofthe fluorescence OFF-time (related to the association rate k_(on)).Extending the imaging/docking duplex from 9 to 10 nucleotides (nt) byadding a single CG base pair, the kinetic OFF-rate is reduced by almostone order of magnitude (8).

FIG. 10A shows a barcode probe that is roughly 50 nucleotide (nt) inlength and can tag a biomolecule using a 21 nt target detection domaint* followed by an approximately 30 nt long “barcode” region with acombination of 8, 9, or 10 nt long binding domain for red, green, orblue imager strands. Here, 8, 9 or 10 nt long docking strands aredisplayed for three colors with a k_(off) of 10, 1 and 0.1 per second,respectively (color rendering not shown). FIG. 10B shows characteristicintensity vs. time traces with increased fluorescence ON-times σ_(b) forthe 9 nt interaction domain compared to the 8 nt interaction domain.FIG. 10C shows stochastic simulations demonstrating that k_(off) valuesof 10, 1 and 0.1 per second can be distinguished.

FIG. 11A shows that in the traditional method of detection, where asingle fluorophore is stably attached to the imaging surface (see FIG.11B(1)), a limited number of photons per “switching” event is emitted(top panel), that extraction of all photons from “replenishable” imagerstrands (see FIG. 11B(2)) leads to higher localization accuracy perswitching event (middle panel), and that a DNA metafluorophore (see FIG.11B(3) and FIG. 11B(4)) yields a significantly larger number of photonsper switching event than the single fluorophore in FIG. 11B(2) (bottom).FIGS. 11B(1)-11B(3) show schematics of current imaging methods andmethods of the present disclosure. FIG. 11B(1) shows a traditionaldetection method (e.g., in STORM), which uses a fluorophore stablyattached to the imaging surface. FIG. 11B(2) shows one embodiment of adetection method of the present disclosure with fluorophores transientlybinding to the imaging surface. FIG. 11B(3) shows a brightmetafluorophore with 8 fluorophores in a compact DNA nanostructure. FIG.11B(4) shows a conditional metafluorophore decorated with both quenchers(dark dots) and fluorophores (stars) that only fluoresces whentransiently bound to the surface. FIG. 11C shows a DNA origami structurewith docking sites arranged in a 4×3 grid, spacing 20 nm. Single sitesare optically localized with an accuracy of approximately 3 nm,currently the highest demonstrated resolution [scale bar: 50 nm]. FIG.11D shows a 280 nm×240 nm DNA nano-rectangle (single-stranded tilestructure (15), 10× larger area than origami) displaying 2000single-stranded docking strands (dots) with 7 or 5 nm spacing used as atest platform for ultra-resolution imaging [scale bar: 100 nm].

FIG. 12A(i) illustrates schematics showing the principle of each stageof drift correction. In each image, black markers and lines indicatesource data, and gray values and curves indicate the calculated driftcorrection. FIG. 12A(ii) shows a schematic drawing of the major type ofdrift markers (e.g., DNA drift markers) used in each stage. FIG. 12B(i)illustrates an example structure showing the imaging quality after eachstage or correction, and FIG. 12B(ii) shows a zoomed image of thecorresponding green rectangle in FIG. 12B(i) at each stage. The scalebars shown in FIGS. 12B(i) and 12B(ii) correspond to 50 nm. FIG. 12C(i)illustrates an example drift trace after each stage of correction, andFIG. 12C(ii) shows a zoomed image of the corresponding rectangle in FIG.12C(i) at each stage. The scale bars in FIG. 12C(i) correspond to x: 500nm, t: 500 s, and the scale bars in FIG. 12C(ii) correspond to x: 10 nm,t: 10 s.

FIG. 13 illustrates a process for performing drift correction inaccordance with some embodiments.

FIG. 14 illustrates a process for performing drift correctioncorresponding to stage 230 of FIG. 13.

FIG. 15 illustrates a process for performing drift correctioncorresponding to stage 240 of FIG. 13.

FIG. 16 illustrates 3D tetrahedrons used as templates for 3D driftcorrection. The four corners are labeled with docking sites. FIG. 16Ashows that the four corners are clearly resolved. FIG. 16B illustratesthe X-Z projection of the structures with a height of ˜85 nm.

FIG. 17 shows an illustrative implementation of computer system 600 thatmay be used in connection with any of the embodiments of the presentdisclosure described herein.

FIGS. 18A-18C illustrate an alternative representation of stages in adrift correction process in accordance with some embodiments of thepresent disclosure. A super-resolved image of a 10 nm-spaced regulargrid on a single-molecule DNA origami nanostructure is shown. The DNAorigami structure was designed to be a 5×8 square lattice of 10 nmspacing both vertically and horizontally. FIG. 18A shows a scatter plotof collected and filtered localizations, represented by crosses. FIG.18B shows a binned 2-D histogram view of the above structure. FIG. 18Cshows a 1-D histogram by projecting all localizations in the rectanglein FIGS. 18A and 18B onto the x-axis, and least square fitting with 8Gaussian components. The fitted Gaussian peaks all have standarddeviation in the range of 1.5-2.4 nm, allowing for 3.5-5.6 nm resolutionin principle; and spacing between neighboring peaks in the range of9.8-11.0 nm, consistent with the DNA origami design. A few (5 in thiscase) spots are missing in the structure because of imperfectincorporation of staples in the assembly reaction, but not missed duringthe super-resolution imaging.

FIGS. 19A and 19B show that RNA aptamers modulate the fluorescence ofGFP-like fluorophores. FIG. 19A shows structures of HBI (green), in thecontext of GFP, and DMHBI. FIG. 19B shows that the 13-2 RNA aptamerenhances the fluorescence of DMHBI by stabilizing a particular moleculararrangement favorable for fluorescence emission. Solutions containingDMHBI, 13-2 RNA, DMHBI with 13-2 RNA, or DMHBI with total HeLa cell RNAwere photographed under illumination with 365 nm of light. The image isa montage obtained under identical image-acquisition conditions. (Imagefrom Paige et al., ref. 19)

FIGS. 20A and 20B show single-molecule fluorescence characterization ofthe DFHBI binding kinetics. FIG. 20A shows a 5′-extended Spinach (green)is immobilized on a

BSA/Biotin-coated glass substrate using a biotinylated DNA capturesequence (labeled with a red dye, e.g. Alexa647; color rendering notshown). FIG. 20B shows a bulk fluorescence measurement of theSpinach-DFHBI before (bottom line) and after (top line) addition of theaptamer shows that the DFHBI binding activity is well maintained afterthe addition an extension to Spinach required for immobilizing to theglass surface in FIG. 20A.

FIGS. 21A and 21B show benchmarking Spinach-PAINT performance. FIG. 20Ashows a six-helix DNA origami structure used for placing two Spinachmolecules in a defined distance. FIG. 20B shows a simulatedrepresentation of a super-resolved reconstruction using DNA-PAINT tolocalize the DNA structure (P) and Spinach-PAINT to localize the Spinachmolecules in three different distances.

FIG. 22 shows Spinach-based sensors. The allosteric variant of theSpinach-based sensor (left) comprises the Spinach domain (black), thetransducer module (medium gray), and the recognition module (lightgray). In the absence of the target molecule, the transducer module isin a primarily unstructured state, which prevents the stabilization ofthe Spinach structure needed for activation of DFHBI. Upon binding ofthe target molecule, the transducer module forms a duplex, leading tostructural rigidification of the Spinach module, and activation of DFHBIfluorescence (ref. 24).

FIGS. 23A and 23B show examples of in vitro and in situ Exchange-PAINTchambers.

DESCRIPTION OF THE INVENTION

The present disclosure provides, inter alia, methods, compositions andkits for multiplexed imaging, for example, in a cellular environmentusing nucleic acid-based imaging probes (e.g., DNA-based imagingprobes). The methods, compositions and kits for multiplexed fluorescenceimaging are not limited by the degree of resolution attained. Thus, themethods, compositions and kits as provided herein may be used forimaging, generally.

In some aspects, the present disclosure further provides, inter alia,methods, compositions and kits for multiplexed super-resolutionfluorescence imaging, for example, in a cellular environment usingnucleic acid-based imaging probes (e.g., DNA-based imaging probes). Asused herein, “super-resolution” imaging refers to the process ofcombining a set of low resolution images of the same area to obtain asingle image of higher resolution. Many aspects of the presentdisclosure may be used to “switch” targets (e.g., biomolecules) ofinterest between fluorescent ON- and OFF-states to permit consecutive,or in some instances simultaneous, localization of individual targets. Afluorescent “ON” state is a state in which fluorescence is emitted. Afluorescent “OFF” state is a state in which fluorescence is not emitted.Switching between the two states is achieved, in some embodiments, withdiffusing molecules that are labeled with a detectable label (e.g.,fluorescent molecules) that interact transiently with the targets usingan intermediate moiety that comprises the detectable label (e.g.,fluorescent molecule(s)) and binds to the target. The methods,compositions and kits of the present disclosure are useful, in someaspects, for detecting, identifying and quantifying target targets ofinterest.

Binding partner-nucleic acid conjugates (“BP-NA conjugates”) of thepresent disclosure transiently bind to complementary labeled, optionallyfluorescently labeled, imager strands. As used herein, “bindingpartner-nucleic acid conjugate,” or “BP-NA conjugate,” refers to amolecule linked (e.g., through an N-Hydroxysuccinimide (NHS) linker) toa single-stranded nucleic acid (e.g., DNA) docking strand. The bindingpartner of the conjugate may be any moiety (e.g., antibody or aptamer)that has an affinity for (e.g., binds to) a target, such as abiomolecule (e.g., protein or nucleic acid), of interest. In someembodiments, the binding partner is a protein. BP-NA-conjugates thatcomprise a protein (or peptide) linked to a docking strand may bereferred to herein as “protein-nucleic acid conjugates,” or “protein-NAconjugates.” Examples of proteins for use in the conjugates of thepresent disclosure include, without limitation, antibodies (e.g.,monoclonal monobodies), antigen-binding antibody fragments (e.g., Fabfragments), receptors, peptides and peptide aptamers. Other bindingpartners may be used in accordance with the present disclosure. Forexample, binding partners that bind to targets through electrostatic(e.g., electrostatic particles), hydrophobic or magnetic (e.g., magneticparticles) interactions are contemplated herein.

As used herein, “antibody” includes full-length antibodies and anyantigen binding fragment (e.g., “antigen-binding portion”) or singlechain thereof. The term “antibody” includes, without limitation, aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Antibodies may be polyclonal or monoclonal; xenogeneic,allogeneic, or syngeneic; or modified forms thereof (e.g., humanized,chimeric).

As used herein, “antigen-binding portion” of an antibody, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen. The antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(H), V_(L), C_(L) and C_(H1) domains; (ii) a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(H) and V_(L)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341:544 546, 1989), which consists of a V_(H) domain; and (vi) anisolated complementarity determining region (CDR) or (vii) a combinationof two or more isolated CDRs, which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(H) and V_(L), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(H) and V_(L)regions pair to form monovalent molecules (known as single chain Fv(scFv); see, e.g., Bird et al. Science 242:423 426, 1988; and Huston etal. Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single chainantibodies are also encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

As used herein, “receptors” refer to cellular-derived molecules (e.g.,proteins) that bind to ligands such as, for example, peptides or smallmolecules (e.g., low molecular weight (<900 Daltons) organic orinorganic compounds).

As used herein, “peptide aptamer” refers to a molecule with a variablepeptide sequence inserted into a constant scaffold protein (see, e.g.,Baines I C, et al. Drug Discov. Today 11:334-341, 2006).

In some embodiments, the molecule of the BP-NA conjugate is a nucleicacid such as, for example, a nucleic acid aptamer. As used herein,“nucleic acid aptamer” refers to a small RNA or DNA molecules that canform secondary and tertiary structures capable of specifically bindingproteins or other cellular targets (see, e.g., Ni X, et al. Curr MedChem. 18(27): 4206-4214, 2011). Thus, in some embodiments, the BP-NAconjugate may be an aptamer-nucleic acid conjugate.

As used herein a “docking strand” refers to a single-stranded nucleicacid (e.g., DNA) that is about 5 nucleotides to about 50 nucleotides inlength (or is 5 nucleotides to 50 nucleotides in length). In someembodiments, a docking strand is about 4 to about 60, about 6nucleotides to about 40 nucleotides, about 7 nucleotides to about 30nucleotides, about 8 to about 20 nucleotides, or about 9 nucleotides toabout 15 nucleotides in length. In some embodiments, a docking strand is(or is about) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, or more nucleotides in length.

A docking strand may have one domain or more than one domain (i.e., aplurality of domains), each domain complementary to a respective imagerstrand. As used herein, a “docking strand domain” refers to a nucleotidesequence of the docking strand that is complementary to a nucleotidesequence of an imager strand. A docking strand, for example, may containone, two three, or more domains, each domain complementary to an imagerstrand. Each complementary imager strand can contain a distinct label(e.g., a red fluorophore, a blue fluorophore, or a green fluorophore),or all complementary imager strands can contain the same label (e.g.,red fluorophores). For example, for a three-domain docking strand, thestrand may contain a first domain complementary to an imager strandlabeled with a red fluorophore, a second domain complementary to animager strand labeled with a blue fluorophore, and a third domaincomplementary to an imager strand labeled with a green fluorophore.Alternatively, each of three docking domains may be complementary toimager strands labeled with a red fluorophore. In some embodiments, adocking strand has at least 2, at least 3, at least 4, at least 5, ormore domains, each respectively complementary to an imager strand. Insome embodiments, a docking strand has 1 to 5, 1 to 10, 1 to 15, 1 to20, 1 to 25, 1 to 50, or 1 to 100 domains, each respectivelycomplementary to an imager strand.

As used herein, an “imager strand” is a single-stranded nucleic acid(e.g., DNA) that is about 4 to about 30 nucleotides, about 5 to about 18nucleotides, about 6 to about 15 nucleotides, about 7 to about 12nucleotides, or about 8 to 10 nucleotides in length and isfluorescently-labeled. In some embodiments, the imager strand may be (ormay be about) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. Animager strand of the present disclosure is complementary to andtransiently binds to a docking strand. Two nucleic acids or nucleic aciddomains are “complementary” to one another if they base-pair, or bind,with each other to form a double-stranded nucleic acid molecule viaWatson-Crick interactions. As used herein, “binding” refers to anassociation between at least two molecules due to, for example,electrostatic, hydrophobic, ionic and/or hydrogen-bond interactionsunder physiological conditions. An imager strand is considered to“transiently bind” to a docking strand if it binds to a complementaryregion of a docking strand and then disassociates (unbinds) from thedocking strand within a short period of time, for example, at roomtemperature. In some embodiments, an imager strand remains bound to adocking strand for about 0.1 to about 10, or about 0.1 to about 5seconds. For example, an imager strand may remain bound to a dockingstrand for about 0.1, about 1, about 5 or about 10 seconds.

Imager strands of the present disclosure may be labeled with adetectable label (e.g., a fluorescent label, and thus are considered“fluorescently labeled”). For example, in some embodiments, an imagerstrand may comprise at least one (i.e., one or more) fluorophore.Examples of fluorophores for use in accordance with the presentdisclosure include, without limitation, xanthene derivatives (e.g.,fluorescein, rhodamine, Oregon green, eosin and Texas red), cyaninederivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine and merocyanine), naphthalene derivatives (e.g., dansyland prodan derivatives), coumarin derivatives, oxadiazole derivatives(e.g., pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole), pyrenederivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red,Nile blue, cresyl violet and oxazine 170), acridine derivatives (e.g.,proflavin, acridine orange and acridine yellow), arylmethine derivatives(e.g., auramine, crystal violet and malachite green), and tetrapyrrolederivatives (e.g., porphin, phthalocyanine and bilirubin). Otherdetectable labels may be used in accordance with the present disclosure,such us, for example, gold nanoparticles or other detectable particlesor moieties.

As used herein, “spectrally distinct” molecules of the presentdisclosure (e.g., conjugates and/or imager strands) refer to moleculeswith labels (e.g., fluorophores) of different spectral signal orwavelength. For example, an imager strand labeled with a Cy2 fluorophoreemits a signal at a wavelength of light of about 510 nm, while an imagerstrand labeled with a Cy5 fluorophore emits a signal at a wavelength oflight of about 670 nm. Thus, the Cy2-labeled imager strand is consideredherein to be spectrally distinct from the Cy5-labeled imager strand.Conversely, “spectrally indistinct” molecules of the present disclosureherein refer to molecules with labels having the same spectral signal orwavelength—that is, the emission wavelength of the labels cannot be usedto distinguish between two spectrally indistinct fluorescently labeledmolecules (e.g., because the wavelengths are the same or closetogether).

The BP-NA conjugates (e.g., protein-nucleic acid conjugates) of thepresent disclosure may, in some embodiments, comprise an intermediatelinker that links (e.g., covalently or non-covalently) the molecule to adocking strand. The intermediate linker may comprise biotin and/orstreptavidin. For example, in some embodiments, an antibody and adocking strand may each be biotinylated (i.e., linked to at least onebiotin molecule) and linked to each other through biotin binding to anintermediate streptavidin molecule, as shown in FIG. 2. Otherintermediate linkers may be used in accordance with the presentdisclosure. In some embodiments, such as those where the molecule is anucleic acid, an intermediate linker may not be required. For example,the docking strand of a BP-NA conjugate may be an extension (e.g., 5′ or3′ extension) of a nucleic acid molecule such as, for example, a nucleicacid aptamer.

Pluralities of BP-NA conjugates (e.g., protein-nucleic acid conjugates)and imager strands are provided herein. A plurality may be a populationof the same species or distinct species. A plurality of BP-NA conjugatesof the same species may comprise conjugates that all bind to the sametarget (e.g., biomolecule) (e.g., the same epitope or region/domain).Conversely, a plurality of BP-NA conjugates of distinct species maycomprise conjugates, or subsets of conjugates, each conjugate or subsetof conjugates binding to a distinct epitope on the same target or to adistinct target. A plurality of imager strands of the same species maycomprise imager strands with the same nucleotide sequence and the samefluorescent label (e.g., Cy2, Cy3 or Cy4). Conversely, a plurality ofimager strands of distinct species may comprise imager strands withdistinct nucleotide sequences (e.g., DNA sequences) and distinctfluorescent labels (e.g., Cy2, Cy3 or Cy4) or with distinct nucleotidesequences and the same fluorescent (e.g., all Cy2). The number ofdistinct species in a given plurality of BP-NA conjugates is limited bythe number of binding partners (e.g., antibodies) and the number ofdocking strands of different nucleotide sequence (and thus complementaryimager strands). In some embodiments, a plurality of BP-NA conjugates(e.g., protein-nucleic acid conjugates) comprises at least 10, 50, 100,500, 1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹ BP-NA conjugates. Likewise, in some embodiments, aplurality of fluorescently-labeled imager strands comprises at least 10,50, 100, 500, 1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ fluorescently-labeled imager strands. In someembodiments, a plurality may contain 1 to about 200 or more distinctspecies of BP-NA conjugates and/or imager strands. For example, aplurality may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200 or more distinct species. In some embodiments, a pluralitymay contain less than about 5 to about 200 distinct species of BP-NAconjugates and/or imager strands. For example, a plurality may containless than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 distinct species.

The present disclosure also contemplates docking strands that can binddirectly to a target. For example, as shown in FIG. 8A, a docking strandmay contain, in additional to imager-binding domain(s) (e.g., one, two,three, or more, with the same or distinct fluorophores), a target domainthat is complementary to and binds to a target, such as, for example,mRNA or other nucleic acid.

Methods

Methods provided herein are based, in part, on the programmability ofnucleic acid docking strands and imager strands. That is, for example,docking strands and imager strands can be designed such that they bindto each other under certain conditions for a certain period of time.This programmability permits transient binding of imager strands todocking strands, as provided herein. Generally, the methods providedherein are directed to identifying one or more target(s) (e.g.,biomolecule(s) such as a protein or nucleic acid) in a particular sample(e.g., biological sample). In some instances, whether or not one or moretarget(s) is present in sample is unknown. Thus, methods of the presentdisclosure may be used to determine the presence or absence of one ormore target(s) in a sample suspected of containing the target(s). In anyone of the aspects and embodiments provided herein, a sample may containor may be suspected of containing one or more target(s).

Methods provided herein can also be used to identify the absolutequantity of a single target (e.g., such as, for example, a particularprotein), or the quantity of a single target relative to one or moreother targets.

Further, methods provided herein may be used to identify the location ofa target within a sample or relative to other targets in the sample.

Methods provided herein may comprise, in some embodiments, contacting asample with (a) at least one BP-NA conjugate (e.g., protein-nucleic acidconjugate) that comprises a binding partner linked to a docking strandand (b) at least one labeled, optionally fluorescently labeled, imagerstrand that is complementary to and transiently binds to the dockingstrand of the at least one BP-NA conjugate, and then determining whetherthe at least one BP-NA conjugate binds to at least one target (such as abiomolecule target) in the sample. In some embodiments, the determiningstep comprises imaging (e.g., with time-lapsed fluorescent microscopytechniques) transient binding of the at least one labeled, optionallyfluorescently labeled, imager strand to the docking strand of the atleast one BP-NA conjugate.

Other methods provided herein may comprise, in some embodiments,contacting a sample with (a) at least two BP-NA conjugates, eachcomprising a binding partner linked to a docking strand, and (b) atleast two labeled, optionally spectrally distinct, fluorescentlylabeled, imager strands that are complementary to and transiently bindto respective docking strands of the at least two different BP-NAconjugates, and then determining whether the at least two BP-NAconjugates bind to at least one, or at least two, targets (such asbiomolecule targets) in the sample. Binding of the BP-NA conjugates torespective targets can be determined by imaging transient binding of oneof the at least two labeled, optionally spectrally distinct,fluorescently labeled, imager strands to a docking strand of one of theat least two BP-NA conjugates to produce a first image, and then imagingtransient binding of another of the at least two labeled, optionallyspectrally distinct, fluorescently labeled, imager strands to a dockingstrand of another of the at least two BP-NA conjugates to produce asecond image. In some embodiments, the methods further comprisecombining the first image and the second image to produce a compositeimage of signals (e.g., fluorescent signals), wherein the signals (e.g.,fluorescent signals) of the composite image are representative of the atleast two targets. As used herein, a “composite image” refers to asingle image produced by combining (e.g., overlaying) multiple images ofthe same (or substantially similar) area. A composite image may also bereferred to as a super-resolution image, as described elsewhere herein.

FIG. 3 demonstrates one embodiment of the present disclosure in whichtwo distinct species of BP-NA conjugates (e.g., antibody-nucleic acidconjugates) are used to label biomolecules in a fixed HeLa cell sample.One species of antibody-nucleic acid conjugate comprises an antibodythat recognizes and binds to an epitope on mitochondria. Themitochondrial specific antibody is linked to a docking strand with asequence complementary to a Cy3b-labled imager strand. The other speciesof antibody-nucleic acid conjugate comprises an antibody that recognizesand binds to an epitope on microtubules. The microtubule specificantibody is linked to a docking strand with a sequence complementary toan ATTO655-labeled imager strand. Two spectrally distinct species ofimager strands are then introduced at the same time: one species islabeled with Cy3b and is complementary to the docking strand that islinked to the mitochondrial specific antibody, and the other species islabeled with ATTO655 and is complementary to the docking strand that islinked to the microtubule specific antibody. While both the Cy3b-labledimager strand and the ATTO655-labled imager strand are present at thesame time in solution with the sample, imaging is carried oursequentially in Cy3b and ATTO655 channels.

Yet other methods provided herein may comprise, in some embodiments,contacting a sample with (a) at least two BP-NA conjugates, eachcomprising a protein linked to a docking strand and (b) at least twospectrally indistinct (e.g., labeled with the same fluorophore)fluorescently-labeled imager strands that are complementary to andtransiently bind to respective docking strands of the at least two BP-NAconjugates, and then determining whether the at last two BP-NAconjugates bind to at least two targets (e.g., biomolecule targets) inthe sample. In some embodiments, the methods comprise, in the followingordered steps, contacting the sample with a first BP-NA conjugate and atleast one other BP-NA conjugate, contacting the sample with a firstfluorescently-labeled imager strand that is complementary to andtransiently binds to the docking strand of the first BP-NA conjugate,determining whether the first BP-NA conjugate binds to a first target,removing the first fluorescently-labeled imager strand, contacting thesample with at least one other fluorescently-labeled imager strand thatis complementary to and transiently binds to the docking strand of theat least one other BP-NA conjugate, and determining whether the at leastone other BP-NA conjugate binds to at least one other target.

Alternatively, in other embodiments, methods comprise, in the followingordered steps, contacting the sample with a first BP-NA conjugate,contacting the sample with a first fluorescently-labeled imager strandthat is complementary to and transiently binds to the docking strand ofthe first BP-NA conjugate, determining whether the first BP-NA conjugatebinds to a first target (e.g., biomolecule), removing the firstfluorescently-labeled imager strand, contacting the sample with at leastone other BP-NA conjugate, contacting the sample with at least one otherfluorescently-labeled imager strand that is complementary to andtransiently binds to the docking strand of the at least one other BP-NAconjugate, and determining whether the at least one other BP-NAconjugate binds to at least one other target.

In some embodiments, the first determining step comprises imagingtransient binding of the first fluorescently-labeled imager strand tothe docking strand of the first BP-NA conjugate to produce a firstimage, and the second determining step comprises imaging transientbinding of the at least one other fluorescently-labeled imager strand tothe docking strand of the at least one other BP-NA conjugate to producea second image. In some embodiments, the methods further compriseassigning a pseudo-color to the fluorescent signal in the first image,and assigning at least one other pseudo-color to the fluorescent signalin the second image. Further still, in some embodiments, the methodscomprise combining the first image and the second image to produce acomposite image of the pseudo-colored signals, wherein thepseudo-colored signals of the composite image are representative of theat least two targets (e.g., biomolecule targets). As illustrated in FIG.4A, step [1], three distinct species of docking strands (a,b,c) labelthe surface of a grid (chosen for illustrative purposes). In step [2],multiple copies of the imager strand a* are introduced, and pointslabeled with docking strands a are imaged. In step [3], copies of theimager strand a* are flushed away, and imager strand b* is introduced toimage the b labeled points. In step [4], c labeled points are imaged inthe same manner. In step [5], images from steps [2-4] are assignedartificial pseudo-colors (e.g., using a software program) and combinedto create the final composite image. All imager strands may be labeledwith the same fluorophore—that is, the imager strands are spectrallyindistinct. In some embodiments, the docking strands are linked tobinding partners (e.g., proteins such as antibodies, or nucleic acidssuch as DNA or nucleic acid aptamers).

An advantage of the methods of the present disclosure is thatpartitioning and sequential imaging can be used to obtain multiplexedsuper-resolved images of up to hundreds of different species using onlya single optimized fluorescent dye. Using these methods, the number ofdistinct nucleotide sequences (e.g., DNA sequences), as opposed to thenumber of spectrally distinct dyes, limits the multiplexing capability.In some methods of the present disclosure, for example, those that usean imager strand with a length of 9 nucleotides, there are severalhundred species within tight bounds for binding kinetics that may beused for a single sample, representing a tremendous increase inmultiplexing compared to direct “traditional” imaging approaches.

FIG. 5A illustrates another embodiment of the present disclosure usingspectrally indistinct imager strands. A single DNA nanostructuredisplays four distinct species of docking strands (optionally linked toprotein binding partners or nucleic acid binding partners) designed toresemble the digits from 0 to 3, respectively. Imaging is performedsequentially using a simple flow chamber setup, first flushing influorescently-labeled imager strands complementary to the dockingstrands of the number 0, and then exchanging the solution forfluorescently-labeled imager strands with a sequence complementary todocking strands of the number 1, and so forth. The resulting images havebeen pseudo-colored to represent the respective imaging cycles. As usedherein, an “imaging cycle,” or “imaging round” refers to the process ofintroducing fluorescently-labeled imager strands complementary todocking strands under conditions that allow the imager strand to bind tothe docking strand, even if such binding is transient, and obtaining animage (or imaging an area).

Aspects of the present disclosure contemplate multiplex detection usingmulti-domain docking strands (e.g., docking strands with more than onedomain), as described above. For illustrative purposes, the followingembodiments are described in terms of a docking strand binding to atarget, e.g., without an intermediate binding partner. It should beunderstood, however, that multi-domain docking strands may be linked toa binding partner (e.g., of a BP-NA conjugate, as provided herein.

In some embodiments, methods comprise contacting one or more target(s)with one or more docking strands, each containing two or more bindingdomains. In other embodiments, methods comprise contacting one or moretarget(s) with two or more docking strands, each containing one bindingdomain. The docking strand domains may have orthogonal sequences. In theexamples that follow, all three targets (protein #1-#3) are present inthe sample.

Detection Based on Spectral Resolution. An exemplary multiplexed targetdetection method follows. A sample contains, or is suspected ofcontaining, three target species—protein #1, protein #2, and protein #3.Three docking strands are designed such that: the first, containingimager binding domain A, binds to protein #1; the second, containingimager binding domain B, binds to protein #2; and the third, containingimager binding domains A and B, binds to protein #3. Complementaryimager strand A′ binds to imager binding domain A of a docking strandand is labeled with a blue fluorophore, and imager strand B′ binds toimager binding domain B of a docking strand and is labeled with a redfluorophore. The sample is first contacted with the docking strands, andsubsequently contacted with the imager strands A′ and B′. The sample isthen imaged. The sample containing the docking strands and the imagerstrands is first imaged under conditions that detect the bluefluorophore. Imaging the blue fluorophore detects protein #1 and protein#3, each bound by an imager strand labeled with the blue fluorophore.Imaging the red fluorophore detects protein #2 and protein #3, eachbound by an imager strand labeled with the red fluorophore. Overlappingimages of the red and blue fluorophores detects protein #3 only, theonly protein bound by an imager strand labeled with a red fluorophoreand an imager strand labeled with a blue fluorophore. Thus, theidentification and location of proteins #1-#3 are identified by theoverlay of images respectively detecting the red and blue fluorophores.

Detection Based on Exchange of Imager Strands. Another exemplarymultiplexed target detection method follows: A sample contains, or issuspected of containing, three target species—protein #1, protein #2,and protein #3. Three docking strands are designed such that: the first,containing imager binding domain A, binds to protein #1; the second,containing imager binding domain B, binds to protein #2; and the third,containing imager binding domains A and B, binds to protein #3.Complementary imager strand A′ binds to imager binding domain A of adocking strand and is labeled with a blue fluorophore, and imager strandB′ binds to imager binding domain B of a docking strand and is alsolabeled with a blue fluorophore. The sample is first contacted with thedocking strands, and subsequently contacted with imager strands A′. Thesample is then imaged under conditions that detect the blue fluorophore.Imaging the blue fluorophore detects protein #1 and protein #3, eachbound by imager strand A′ labeled with the blue fluorophore. The sampleis then washed to remove imager strands A′. Next, the sample iscontacted with imager strands B′. The sample is then imaged again underconditions that detect the blue fluorophore. Imaging the bluefluorophore now detects protein #2 and protein #3, each bound by imagerstrand B′ labeled with the blue fluorophore. Overlapping images of theblue fluorophores (e.g., resulting in a stronger signal relative tonon-overlapping fluorophores) detects protein #3 only, the only proteinbound by two imager strands labeled with a blue fluorophore. Thus, theidentification and location of proteins #1-#3 are identified by theoverlay of images detecting the blue fluorophores and is based on signalintensity.

Detection Based on a Combination of Spectral and Exchange Detection. Yetanother exemplary multiplexed target detection method follows: A samplecontains, or is suspected of containing, fifteen target species. Thedocking strands are designed such that each target species binds to adocking strand, each docking strand containing a single distinct domainor a distinct combination of domains A-D (e.g., A, or A and B (i.e.,A/B), or A/C, or A/D, or A/B/C, or A/B/D, or A/C/D, or A/B/C/D, or B, orB/C, or B/D, or B/C/D, or C, or C/D, or D). The imager strands aredivided into two sets: the first set (set #1) contains imager strand A′labeled by a red fluorophore and imager strand B′ labeled by a bluefluorophore; the second set contains imager strand C′ labeled with a redfluorophore and imager strand D′ labeled with a blue fluorophore. Thesample is first contacted with the docking strands, and subsequentlycontacted with imager strand set #1. The sample is then imaged underconditions that detect blue and red fluorophores. Targets bound byimager strands A′ will be detected red and targets bound by imagerstrands B′ will be detected blue. Thus, all target species with dockingdomains A and B will be detected in a first image or first set ofimages. The sample is then washed to remove imager strand set #1. Next,the sample is contacted with imager strand set #2. The sample is thenimaged again under conditions that detect blue and red fluorophores.Targets bound by imager strands C′ will be detected red and targetsbound by imager strands D′ will be detected blue. Thus, all targetspecies with docking domains C and D will be detected in a second imageor second set of images. By combining all images collected, each of the15 target species can be identified using only four imager strands andtwo fluorophores. It should be understood that more than four imagerstrands can be used as well as more than two fluorophores, depending on,for example, the number of targets.

Detection Based on Duration of Transient Binding. In some embodiments,the disclosure contemplates contacting target species with differentdocking strands domain sequence and different lengths of thosesequences. The length of a docking strand imager binding domain affectsthe duration of transient binding to an imager strand. Docking strandswith longer binding domains bind to respectively complementary imagerstrands for longer durations relative to shorter binding domains. In thefollowing exemplary embodiment, a sample contains, or is suspected ofcontaining, four target species. Four docking strands are designed suchthat: the first, containing binding domain A of 10 nucleotides in length(A10), binds to protein #1; the second, containing imager binding domainA10 and imager binding domain B of 8 nucleotides in length (B8), bindsto protein #2; the third, containing imager binding domain A of 8nucleotides in length (A8) and imager binding domain B of 10 nucleotidesin length (B10), binds to protein #3; and four, containing imager strandbinding domain B10. Imager strand A′ is 10 nucleotides in length, bindsto both A8 and A10, and is labeled with a blue fluorophore. Imagerstrand B′ is 10 nucleotides in length, binds to both B8 and B10, and islabeled with a red fluorophore. The sample is first contacted with thedocking strands, and subsequently contacted with imager strands A′ andB′. The sample is then imaged under conditions that detect the bluefluorophore. Imaging the blue fluorophore detects protein #3 and protein#4 with a longer bound time (i.e., time of binding between imager strandand docking strand) and protein #2 with a shorter bound time. Imagingthe red fluorophore detects protein #1 and protein #2 with a longerbound time and protein #3 with a shorter bound time. Overlapping imagesof the blue and red fluorophores detects each of the four proteintargets.

The present disclosure also contemplates combining multiplexed detectionbased on spectral resolution and duration, exchange and duration, andspectral resolution, exchange and duration.

A “sample” may comprise cells (or a cell), tissue, or bodily fluid suchas blood (serum and/or plasma), urine, semen, lymphatic fluid,cerebrospinal fluid or amniotic fluid. A sample may be obtained from (orderived from) any source including, without limitation, humans, animals,bacteria, viruses, microbes and plants. In some embodiments, a sample isa cell lysate or a tissue lysate. A sample may also contain mixtures ofmaterial from one source or different sources. A sample may be a spatialarea or volume (e.g., a grid on an array, or a well in a plate or dish).A sample, in some embodiments, includes target(s), BP-NA conjugate(s)and imager strand(s).

A “target” is any moiety that one wishes to observe or quantitate andfor which a binding partner exists. A target, in some embodiments, maybe non-naturally occurring. The target, in some embodiments, may be abiomolecule. As used herein, a “biomolecule” is any molecule that isproduced by a living organism, including large macromolecules such asproteins, polysaccharides, lipids and nucleic acids (e.g., DNA and RNAsuch as mRNA), as well as small molecules such as primary metabolites,secondary metabolites, and natural products. Examples of biomoleculesinclude, without limitation, DNA, RNA, cDNA, or the DNA product of RNAsubjected to reverse transcription, A23187 (Calcimycin, CalciumIonophore), Abamectine, Abietic acid, Acetic acid, Acetylcholine, Actin,Actinomycin D, Adenosine, Adenosine diphosphate (ADP), Adenosinemonophosphate (AMP), Adenosine triphosphate (ATP), Adenylate cyclase,Adonitol, Adrenaline, epinephrine, Adrenocorticotropic hormone (ACTH),Aequorin, Aflatoxin, Agar, Alamethicin, Alanine, Albumins, Aldosterone,Aleurone, Alpha-amanitin, Allantoin, Allethrin, α-Amanatin, Amino acid,Amylase, Anabolic steroid, Anethole, Angiotensinogen, Anisomycin,Antidiuretic hormone (ADH), Arabinose, Arginine, Ascomycin, Ascorbicacid (vitamin C), Asparagine, Aspartic acid, Asymmetricdimethylarginine, Atrial-natriuretic peptide (ANP), Auxin, Avidin,Azadirachtin A—C35H44O16, Bacteriocin, Beauvericin, Bicuculline,Bilirubin, Biopolymer, Biotin (Vitamin H), Brefeldin A, Brassinolide,Brucine, Cadaverine, Caffeine, Calciferol (Vitamin D), Calcitonin,Calmodulin, Calmodulin, Calreticulin, Camphor—(C10H160), Cannabinol,Capsaicin, Carbohydrase, Carbohydrate, Carnitine, Carrageenan, Casein,Caspase, Cellulase, Cellulose—(C6H10O5), Cerulenin, Cetrimonium bromide(Cetrimide)—C 19H42BrN, Chelerythrine, Chromomycin A3, Chaparonin,Chitin, α-Chloralose, Chlorophyll, Cholecystokinin (CCK), Cholesterol,Choline, Chondroitin sulfate, Cinnamaldehyde, Citral, Citric acid,Citrinin, Citronellal, Citronellol, Citrulline, Cobalamin (vitamin B12),Coenzyme, Coenzyme Q, Colchicine, Collagen, Coniine, Corticosteroid,Corticosterone, Corticotropin-releasing hormone (CRH), Cortisol,Creatine, Creatine kinase, Crystallin, α-Cyclodextrin, Cyclodextringlycosyltransferase, Cyclopamine, Cyclopiazonic acid, Cysteine, Cystine,Cytidine, Cytochalasin, Cytochalasin E, Cytochrome, Cytochrome C,Cytochrome c oxidase, Cytochrome c peroxidase, Cytokine,Cytosine—C4H5N3O, Deoxycholic acid, DON (DeoxyNivalenol),Deoxyribofuranose, Deoxyribose, Deoxyribose nucleic acid (DNA), Dextran,Dextrin, DNA, Dopamine, Enzyme, Ephedrine, Epinephrine—C9H13NO3, Erucicacid—CH3(CH2)7CH═CH(CH2)11COOH, Erythritol, Erythropoietin (EPO),Estradiol, Eugenol, Fatty acid, Fibrin, Fibronectin, Folic acid (VitaminM), Follicle stimulating hormone (FSH), Formaldehyde, Formic acid,Formnoci, Fructose, Fumonisin B1, Gamma globulin, Galactose, Gammaglobulin, Gamma-aminobutyric acid, Gamma-butyrolactone,Gamma-hydroxybutyrate (GHB), Gastrin, Gelatin, Geraniol, Globulin,Glucagon, Glucosamine, Glucose—C6H12O6, Glucose oxidase, Gluten,Glutamic acid, Glutamine, Glutathione, Gluten, Glycerin (glycerol),Glycine, Glycogen, Glycolic acid, Glycoprotein, Gonadotropin-releasinghormone (GnRH), Granzyme, Green fluorescent protein, Growth hormone,Growth hormone-releasing hormone (GHRH), GTPase, Guanine, Guanosine,Guanosine triphosphate (+GTP), Haptoglobin, Hematoxylin, Heme,Hemerythrin, Hemocyanin, Hemoglobin, Hemoprotein, Heparan sulfate, Highdensity lipoprotein, HDL, Histamine, Histidine, Histone, Histonemethyltransferase, HLA antigen, Homocysteine, Hormone, human chorionicgonadotropin (hCG), Human growth hormone, Hyaluronate, Hyaluronidase,Hydrogen peroxide, 5-Hydroxymethylcytosine, Hydroxyproline,5-Hydroxytryptamine, Indigo dye, Indole, Inosine, Inositol, Insulin,Insulin-like growth factor, Integral membrane protein, Integrase,Integrin, Intein, Interferon, Inulin, Ionomycin, Ionone, Isoleucine,Iron-sulfur cluster, K252a, K252b, KT5720, KT5823, Keratin, Kinase,Lactase, Lactic acid, Lactose, Lanolin, Lauric acid, Leptin, LeptomycinB, Leucine, Lignin, Limonene, Linalool, Linoleic acid, Linolenic acid,Lipase, Lipid, Lipid anchored protein, Lipoamide, Lipoprotein, Lowdensity lipoprotein, LDL, Luteinizing hormone (LH), Lycopene, Lysine,Lysozyme, Malic acid, Maltose, Melatonin, Membrane protein,Metalloprotein, Metallothionein, Methionine, Mimosine, Mithramycin A,Mitomycin C, Monomer, Mycophenolic acid, Myoglobin, Myosin, Naturalphenols, Nucleic Acid, Ochratoxin A, Oestrogens, Oligopeptide,Oligomycin, Orcin, Orexin, Ornithine, Oxalic acid, Oxidase, Oxytocin,p53, PABA, Paclitaxel, Palmitic acid, Pantothenic acid (vitamin B5),parathyroid hormone (PTH), Paraprotein, Pardaxin, Parthenolide, Patulin,Paxilline, Penicillic acid, Penicillin, Penitrem A, Peptidase, Pepsin,Peptide, Perimycin, Peripheral membrane protein, Perosamine,Phenethylamine, Phenylalanine, Phosphagen, phosphatase, Phospholipid,Phenylalanine, Phytic acid, Plant hormones, Polypeptide, Polyphenols,Polysaccharides, Porphyrin, Prion, Progesterone, Prolactin (PRL),Proline, Propionic acid, Protamine, Protease, Protein, Proteinoid,Putrescine, Pyrethrin, Pyridoxine or pyridoxamine (Vitamin B6),Pyrrolysine, Pyruvic acid, Quinone, Radicicol, Raffinose, Renin,Retinene, Retinol (Vitamin A), Rhodopsin (visual purple), Riboflavin(vitamin B2), Ribofuranose, Ribose, Ribozyme, Ricin, RNA—Ribonucleicacid, RuBisCO, Safrole, Salicylaldehyde, Salicylic acid,Salvinorin-A—C23H2808, Saponin, Secretin, Selenocysteine,Selenomethionine, Selenoprotein, Serine, Serine kinase, Serotonin,Skatole, Signal recognition particle, Somatostatin, Sorbic acid,Squalene, Staurosporin, Stearic acid, Sterigmatocystin, Sterol,Strychnine, Sucrose (sugar), Sugars (in general), superoxide, T2 Toxin,Tannic acid, Tannin, Tartaric acid, Taurine, Tetrodotoxin, Thaumatin,Topoisomerase, Tyrosine kinase, Taurine, Testosterone,Tetrahydrocannabinol (THC), Tetrodotoxin, Thapsigargin, Thaumatin,Thiamine (vitamin B1)—C12H17ClN4OS.HCl, Threonine, Thrombopoietin,Thymidine, Thymine, Triacsin C, Thyroid-stimulating hormone (TSH),Thyrotropin-releasing hormone (TRH), Thyroxine (T4), Tocopherol (VitaminE), Topoisomerase, Triiodothyronine (T3), Transmembrane receptor,Trichostatin A, Trophic hormone, Trypsin, Tryptophan, Tubulin,Tunicamycin, Tyrosine, Ubiquitin, Uracil, Urea, Urease, Uricacid—C5H4N4O3, Uridine, Valine, Valinomycin, Vanabins, Vasopressin,Verruculogen, Vitamins (in general), Vitamin A (retinol), Vitamin B,Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin ornicotinic acid), Vitamin B4 (adenine), Vitamin B5 (pantothenic acid),Vitamin B6 (pyridoxine or pyridoxamine), Vitamin B12 (cobalamin),Vitamin C (ascorbic acid), Vitamin D (calciferol), Vitamin E(tocopherol), Vitamin F, Vitamin H (biotin), Vitamin K (naphthoquinone),Vitamin M (folic acid), Wortmannin and Xylose.

In some embodiments, a target may be a protein target such as, forexample, proteins of a cellular environment (e.g., intracellular ormembrane proteins). Examples of proteins include, without limitation,fibrous proteins such as cytoskeletal proteins (e.g., actin, arp2/3,coronin, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau,titin, tropomyosin, tubulin and collagen) and extracellular matrixproteins (e.g., collagen, elastin, f-spondin, pikachurin, andfibronectin); globular proteins such as plasma proteins (e.g., serumamyloid P component and serum albumin), coagulation factors (e.g.,complement proteins,C1-inhibitor and C3-convertase, Factor VIII, FactorXIII, fibrin, Protein C, Protein S, Protein Z, Protein Z-relatedprotease inhibitor, thrombin, Von Willebrand Factor) and acute phaseproteins such as C-reactive protein; hemoproteins; cell adhesionproteins (e.g., cadherin, ependymin, integrin, Ncam and selectin);transmembrane transport proteins (e.g., CFTR, glycophorin D andscramblase) such as ion channels (e.g., ligand-gated ion channels suchnicotinic acetylcholine receptors and GABAa receptors, and voltage-gatedion channels such as potassium, calcium and sodium channels),synport/antiport proteins (e.g., glucose transporter); hormones andgrowth factors (e.g., epidermal growth factor (EGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), peptidehormones such as insulin, insulin-like growth factor and oxytocin, andsteroid hormones such as androgens, estrogens and progesterones);receptors such as transmembrane receptors (e.g., G-protein-coupledreceptor, rhodopsin) and intracellular receptors (e.g., estrogenreceptor); DNA-binding proteins (e.g., histones, protamines, CIprotein); transcription regulators (e.g., c-myc, FOXP2, FOXP3, MyoD andP53); immune system proteins (e.g., immunoglobulins, majorhistocompatibility antigens and T cell receptors); nutrientstorage/transport proteins (e.g., ferritin); chaperone proteins; andenzymes.

In some embodiments, a target may be a nucleic acid target such as, forexample, nucleic acids of a cellular environment. As used herein withrespect to targets, docking strands, and imager strands, a “nucleicacid” refers to a polymeric form of nucleotides of any length, such asdeoxyribonucleotides or ribonucleotides, or analogs thereof. Forexample, a nucleic acid may be a DNA, RNA or the DNA product of RNAsubjected to reverse transcription. Non-limiting examples of nucleicacids include coding or non-coding regions of a gene or gene fragment,loci (locus) defined from linkage analysis, exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantnucleic acids, branched nucleic acids, plasmids, vectors, isolated DNAof any sequence, isolated RNA of any sequence, nucleic acid probes, andprimers. Other examples of nucleic acids include, without limitation,cDNA, aptamers, peptide nucleic acids (“PNA”), 2′-5′ DNA (a syntheticmaterial with a shortened backbone that has a base-spacing that matchesthe A conformation of DNA; 2′-5′ DNA will not normally hybridize withDNA in the B form, but it will hybridize readily with RNA), lockednucleic acids (“LNA”), and nucleic acids with modified backbones (e.g.,base- or sugar-modified forms of naturally-occurring nucleic acids). Anucleic acid may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs (“analogous” forms of purines andpyrimidines are well known in the art). If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. A nucleic acid may be a single-stranded, double-stranded,partially single-stranded, or partially double-stranded DNA or RNA.

In some embodiments, a nucleic acid (e.g., a nucleic acid target) isnaturally-occurring. As used herein, a “naturally occurring” refers to anucleic acid that is present in organisms or viruses that exist innature in the absence of human intervention. In some embodiments, anucleic acid naturally occurs in an organism or virus. In someembodiments, a nucleic acid is genomic DNA, messenger RNA, ribosomalRNA, micro-RNA, pre-micro-RNA, pro-micro-RNA, viral DNA, viral RNA orpiwi-RNA. In some embodiments, a nucleic acid target is not a syntheticDNA nanostructure (e.g., two-dimensional (2-D) or three-dimensional(3-D) DNA nanostructure that comprises two or more nucleic acidshybridized to each other by Watson-Crick interactions to form the 2-D or3-D nanostructure).

The nucleic acid docking strands and imager strands described herein canbe any one of the nucleic acids described above (e.g., DNA, RNA,modified nucleic acids, nucleic acid analogues, naturally-occurringnucleic acids, synthetic nucleic acids).

Quantitative Imaging

The present disclosure also provides methods for quantitatingfluorescent moieties or emitters in a dense cluster that cannot bespatially resolved using prior art imaging techniques. Prior to theinvention, no systematic model existed that describes the kinetics ofphotoswitching of fluorescent signals.

Stochastic super-resolution imaging using transient binding of shortoligonucleotides (e.g., imager strands) to their targets offers a uniquepossibility to quantitatively count integer numbers of labeled moleculesin a diffraction-limited area. “Switching” molecules from a fluorescentOFF- to an ON-state in the method of the present disclosure isfacilitated by single-molecule nucleic acid (e.g., DNA) hybridizationevents, which are governed by a very predictable kinetic model with asecond order association rate k_(on) and a first order dissociation ratek_(off):

The kinetic parameters k_(on) and k_(off) are now directly linked tofluorescent ON- and OFF-times (τ_(b) and τ_(d), respectively) depictedin FIG. 7A. The fluorescence ON-time τ_(b) is determined by thedissociation rate k_(off): τ_(b)=1/k_(off), and the fluorescenceOFF-time τ_(d) is determined by the association rate k_(on), theconcentration of imager strands in solution c_(imager), and the numberof observed binding sites bs:

$\tau_{d} = \frac{1}{k_{on} \cdot c_{imager} \cdot {bs}}$

After calibrating k_(on)·c_(imager) using a sample with a known numberof binding sites bs (which can be easily done using, e.g., a DNAnanostructure), the number of binding sites for an unknown molecule orarea can be obtained according to the equation:

${bs} = \frac{1}{k_{on} \cdot c_{imager} \cdot \tau_{d}}$

Accordingly, the quantification of a fluorescence image may be doneautomatically using binding kinetics analysis software. In brief, atypical image is recorded in a time-lapsed fashion (e.g., 15000 frameswith a frame rate of 10 Hz). Fluorescence spot detection and fitting(e.g., Gaussian fitting, Centroid fitting, or Bessel fitting) isperformed on the diffraction-limited image, and thus a super-resolvedimage is obtained. In the next step, a calibration marker is selected(e.g., a DNA origami structure with a defined number of spots as in FIG.7C). The software automatically calculates the fluorescence dark timeτ_(d) by fitting the OFF-time distribution to a cumulative distributionfunction. Using the equations described above, the product ofk_(on)·c_(imager) can be calculated. This product is used to calculatethe number of docking sites, and thus targets in the imaged area.

In some embodiments, the selection of areas of interest in the resolved(e.g., super-resolved) imaged can be performed automatically by applyinga second spot detection step, e.g., to calculate the number of targetsin a cluster.

Thus, in some embodiments, the methods of the present disclosurecomprise providing a sample that comprises targets transiently bounddirectly or indirectly to fluorescently-labeled imager strands,obtaining a time-lapsed diffraction-limited fluorescence image of thesample, performing fluorescence spot detection and fitting (e.g.,Gaussian fitting, Centroid fitting, or Bessel fitting) on thediffraction-limited image to obtain a high-resolution image of thesample, calibrating k_(on)·c_(imager) using a sample with a known numberof targets, wherein k_(on) is a second order association constant, andc_(imager) is the concentration of fluorescently-labeled imager strandsin the sample, including unbound imager strands, determining variableτ_(d) by fitting the fluorescence OFF-time distribution to a cumulativedistribution function, and determining the number of targets in thesample based on the equation, number oftargets=(k_(on)·c_(imager)·τ_(d))⁻¹.

Some aspects of the present disclosure relate to fitting functions. A“fitting function,” as used herein, refers to a mathematical functionused to fit the intensity profile of molecules. Examples of fittingfunctions for use as provided herein include, without limitation,Gaussian fitting, Centroid fitting, and Bessel fitting. It should beunderstood that while many aspects and embodiments of the presentdisclosure refer to Gaussian fitting, other fitting functions may beused instead of, or in addition to, Gaussian fitting.

Compositions

Provided herein are compositions that comprise at least one or at leasttwo (e.g., a plurality) BP-NA conjugate(s) (e.g., protein-nucleic acidconjugate(s)) of the present disclosure. The BP-NA conjugates may bebound to a target of interest (e.g., biomolecule) and/or transientlybound to a complementary fluorescently-labeled imager strand. Acomposition may comprise a plurality of the same species or distinctspecies of BP-NA conjugates. In some embodiments, a composition maycomprise at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 10⁴,50000, 10⁵, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ BP-NA conjugates. Insome embodiments, a composition may comprise at least 10, 50, 100, 500,1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹ complementary fluorescently-labeled imager strands. In someembodiments, a composition may contain 1 to about 200 or more distinctspecies of BP-NA conjugates and/or imager strands. For example, acomposition may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200 or more distinct species. In some embodiments, acomposition may contain less than about 5 to about 200 distinct speciesof BP-NA conjugates and/or imager strands. For example, a compositionmay contain less than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 distinctspecies.

It should be understood that the number of complementaryfluorescently-labeled imager strands imager stands in a composition maybe less than, equal to or greater than the number of BP-NA conjugates inthe composition.

Kits

The present disclosure further provides kits comprising one or morecomponents as provided herein. The kits may comprise, for example, aBP-NA conjugate and/or a fluorescently-labeled imager strands. The kitsmay also comprise components for producing a BP-NA conjugate or forlabeling an imager strand. For example, the kits may comprise a bindingpartner (e.g., antibody), docking strands and intermediate linkers suchas, for example, biotin and streptavidin molecules, and/or imagerstrands. The kits can be used for any purpose apparent to those of skillin the art, including, those described above.

The kits may include other reagents as well, for example, buffers forperforming hybridization reactions. The kit may also includeinstructions for using the components of the kit, and/or for makingand/or using the BP-NA conjugates and/or labeled imager strands.

In some embodiments, a kit comprises at least one docking strand and atleast one labeled imager strand that is capable of transiently bindingto a docking strand. The docking strands may or may not be conjugated toa binding partner. In some embodiments, the docking strands areconjugated to “generic” non-target-specific affinity molecule (e.g.,biotin or streptavidin), which may be used to link a docking strand tobinding partner chosen by an end user. In some embodiments, the affinitymolecule is a secondary antibody. Thus, in some embodiments, a kitcomprises at least one docking strand, at least one affinity moleculesuch as a secondary antibody, and at least one imager strand.

In some embodiments, a kit comprises (a) at least one docking strandlinked to a binding partner such as a protein (e.g., a protein thatbinds to a target) and (b) at least one (e.g., at least 2, at least 3,at least 4, at least 5, at least 10, at least 100) labeled imager strandthat is capable of transiently binding (e.g., transiently binds) to adocking strand. A docking strand may comprise, for example, at least twodomains or at least three domain, wherein each domain binds to arespective complementary labeled imager strand. The number of labeledimager strands may be, for example, less than, greater than or equal tothe number of docking strands. The binding partner may be a protein suchas, for example, an antibody (e.g., monoclonal antibody), anantigen-binding antibody fragment, or a peptide aptamer. In someembodiments, a kit comprises at least two different binding partners(e.g., proteins), each specific for a different target. A bindingpartner (e.g., protein), in some embodiments, is linked to a dockingstrand through an intermediate linker such as, for example, a linkerthat includes biotin and streptavidin (e.g., abiotin-streptavidin-biotin linker). In some embodiments, a dockingstrand is modified to contain an affinity molecule that can be used tolink the docking strand to a binding partner. In some embodiments, theaffinity molecule is a secondary antibody. An imager strand, in someembodiments, is labeled with at least one fluorescent label (e.g., atleast one fluorophore). In some embodiments, the length of an imagerstrand is 4 to 30 nucleotides, or longer. For example, the length of animager strand may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Insome embodiments, the length of an imager strand is 8 to 10 nucleotides.In some embodiments, a kit comprises at least two imager strands, eachdifferent from one another. In some embodiments, the thermal stabilityof a docking strand transiently bound to its complementary labeledimager strand is within 0.5 kcal/mol of the thermal stability of otherdocking strands transiently bound to their respective labeled imagerstrands.

In some embodiments, a kit comprises (a) at least one docking strandlinked to a monoclonal antibody or an antigen binding fragment thereof(e.g., a monoclonal antibody or an antigen binding fragment thereof thatbinds to a target) and (b) at least one (e.g., at least 2, at least 3,at least 4, at least 5, at least 10, at least 100) labeled imager strandthat is capable of transiently binding (e.g., transiently binds) to adocking strand. A docking strand may comprise, for example, at least twodomains, wherein each domain binds to a respectively complementarylabeled imager strand. The number of labeled imager strands may be, forexample, less than, greater than or equal to the number of dockingstrands. In some embodiments, a kit comprises at least two differentmonoclonal antibodies or antigen binding fragments thereof, eachspecific for a different target. A monoclonal antibody or an antigenbinding fragment thereof, in some embodiments, is linked to a dockingstrand through an intermediate linker that includes biotin andstreptavidin (e.g., a biotin-streptavidin-biotin linker). An imagerstrand, in some embodiments, is labeled with at least one fluorescentlabel (e.g., at least one fluorophore). In some embodiments, the lengthof an imager strand is 4 to 30 nucleotides, or longer. For example, thelength of an imager strand may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides. In some embodiments, the length of an imager strand is 8 to10 nucleotides. In some embodiments, a kit comprises at least two imagerstrands, each different from one another. In some embodiments, thethermal stability of a docking strand transiently bound to acomplementary labeled imager strand is within 0.5 kcal/mol of thethermal stability of other docking strands transiently bound to theirrespective labeled imager strands.

Applications

The BP-NA conjugates (e.g., protein-nucleic acid conjugates orantibody-nucleic acid conjugates) of the present disclosure can be used,inter alia, in any assay in which existing target detection technologiesare used.

Typically assays include detection assays including diagnostic assays,prognostic assays, patient monitoring assays, screening assays,biowarfare assays, forensic analysis assays, prenatal genomic diagnosticassays and the like. The assay may be an in vitro assay or an in vivoassay. The present disclosure provides the advantage that many differenttargets can be analyzed at one time from a single sample using themethods of the present disclosure, even where such targets are spatiallynot resolvable (and thus spatially indistinct) using prior art imagingmethods. This allows, for example, for several diagnostic tests to beperformed on one sample.

The BP-NA conjugates can also be used to simply observe an area orregion.

The methods of the present disclosure may be applied to the analysis ofsamples obtained or derived from a patient so as to determine whether adiseased cell type is present in the sample and/or to stage the disease.For example, a blood sample can be assayed according to any of themethods described herein to determine the presence and/or quantity ofmarkers of a cancerous cell type in the sample, thereby diagnosing orstaging the cancer.

Alternatively, the methods described herein can be used to diagnosepathogen infections, for example infections by intracellular bacteriaand viruses, by determining the presence and/or quantity of markers ofbacterium or virus, respectively, in the sample. Thus, the targetsdetected using the methods, compositions and kits of the presentdisclosure may be either patient markers (such as a cancer marker) ormarkers of infection with a foreign agent, such as bacterial or viralmarkers.

The quantitative imaging methods of the present disclosure may be used,for example, to quantify targets (e.g., target biomolecules) whoseabundance is indicative of a biological state or disease condition(e.g., blood markers that are upregulated or downregulated as a resultof a disease state).

Further, the methods, compositions and kits of the present disclosuremay be used to provide prognostic information that assists indetermining a course of treatment for a patient. For example, the amountof a particular marker for a tumor can be accurately quantified fromeven a small sample from a patient. For certain diseases like breastcancer, overexpression of certain proteins, such as Her2-neu, indicate amore aggressive course of treatment will be needed.

The methods of the present disclosure may also be used for determiningthe effect of a perturbation, including chemical compounds, mutations,temperature changes, growth hormones, growth factors, disease, or achange in culture conditions, on various targes, thereby identifyingtargets whose presence, absence or levels are indicative of a particularbiological states. In some embodiments, the present disclosure is usedto elucidate and discover components and pathways of disease states. Forexample, the comparison of quantities of targets present in a diseasetissue with “normal” tissue allows the elucidation of important targetsinvolved in the disease, thereby identifying targets for thediscovery/screening of new drug candidates that can be used to treatdisease.

The sample being analyzed may be a biological sample, such as blood,sputum, lymph, mucous, stool, urine and the like. The sample may be anenvironmental sample such as a water sample, an air sample, a foodsample and the like. The assay may be carried out with one or morecomponents of the binding reaction immobilized. Thus, the targets or theBP-NA conjugates may be immobilized. The assay may be carried out withone or more components of the binding reaction non-immobilized. Theassays may involve detection of a number of targets in a sample,essentially at the same time, in view of the multiplexing potentialoffered by the BP-NA conjugates and fluorescently-labeled imager strandsof the present disclosure. As an example, an assay may be used to detecta particular cell type (e.g., based on a specific cell surface receptor)and a particular genetic mutation in that particular cell type. In thisway, an end user may be able to determine how many cells of a particulartype carry the mutation of interest, as an example.

Devices

Also provided herein are fluidic chamber devices for liquid handling, asshown in FIGS. 23A and 23B. In some embodiments, the device is apolymer-based (e.g., polydimethylsiloxane (PDMS)) device comprisingfirst and second channels, each connected at one end to a samplechamber. This configuration permits one or more fluid(s) to besequentially administered to the sample at a controlled rate. Forexample, a syringe may be used to administer a first fluid to the samplethrough a first channel of the device. The syringe may then be used toadminister a second fluid, which passes through the first channel of thedevice into the sample chamber, thereby forcing the first fluid out ofthe sample chamber, passing through a second channel and into, forexample, a reservoir connected to the second channel (FIG. 23A). In someembodiments, the device is positioned on a glass slide to permit viewingfrom a microscope objective positioned below the device.

Super Resolution Imaging

Super-resolution imaging with increased spatial resolution (referred toherein as “ultra-resolution” imaging) may be achieved, in someembodiments, by increasing the number of photons per localization eventusing one of two strategies, depicted in FIG. 11A. In the firststrategy, the maximum number of photons from single, replenishablefluorophores is extracted. High laser excitation power, in combinationwith fluorophore stabilization buffers (16,17) may be used to “bleach”transiently bound fluorophores at docking sites (or sites of dockingstrands), thus extracting the maximal number of photons per bindingevent and dye (FIG. 11A). The repetitive binding of imager strandspermits “photobleaching” of every bound strand, thus making maximal useof emitted photons and resulting in a significant increase inlocalization accuracy over traditional imaging techniques. In the secondstrategy, bright metafluorophores are used. A fluorescent DNAnanostructure, or “metafluorophore,” may be constructed by decorating acompact DNA nanostructure with many fluorophores (FIG. 11B3). The sum ofthe individual dye emissions from a metafluorophore are interpreted asoriginating from the same point source, and thus, using themetafluorophore in place of a standard fluorophore (e.g., Cy3) furtherimproves the localization precision. A more advanced version of themetafluorophore with active background suppression is depicted in FIG.11B4. Here, the clam-shell-like structure acts as a conditionalfluorophore that only fluoresces when it is bound to the docking strand.

The present disclosure also provides algorithms used for spot detection,fitting and drift correction, as described below.

Software Algorithm for Drift Correction

Some embodiments are directed to methods and apparatus for correctingdrift in images recorded in a time sequence. A non-limiting applicationof the techniques for performing drift correction, discussed in furtherdetail below, is to correct for drift in molecular scale DNA-basedimaging described herein involving transiently binding between dockingstrands and imaging strands. However, it should be appreciated that thetechniques described herein may alternatively be used to correct fordrift in other imaging applications where one or more transient imagingevents are recorded during a time sequence of images, and embodimentsrelated to drift correction are not limited to molecular scale DNA-basedimaging.

In some embodiments, a DNA nanostructure can be used as a drift marker.Any suitable DNA nanostructure (see, e.g., (Rothemund US-2007/0117109A1), single-stranded tiles (Yin et al., “Programming DNA TubeCircumferences,” Science (2008): 321: 824-826), DNA hairpins (Yin et al.US-2009/0011956 A1; Yin et al., “Programming biomolecular self-assemblypathways,” Nature (2008) 451:318-323) may be used, and may be madeusing, e.g., DNA origami techniques. Drift correction using DNAnanostructure-based drift markers in combination with advanced analysisand post-processing techniques has the advantage of high precisioncorrection, compatibility with long time imaging and simplicity ofimplementation. Conventional nucleic acid-based imaging techniquesincorporating drift markers based on fluorescent beads suffer from thelimited length of imaging time before the beads are bleached; whereasbright field imaging requires specialized equipment, e.g. dual-fieldcamera view.

Drift correction techniques in accordance with some embodimentsdescribed herein may include a plurality of stages, where each of thestages uses a different technique to perform drift correction. In someembodiments, the output from one stage is provided as input to asubsequent stage for additional drift correction processing. In a firststage, a coarse drift correction is performed by comparing localizationsfrom neighboring frames. In a second stage, a single drift marker isselected and its time trace is used as a different coarse correction. Ina third stage, a group of drift markers is selected, eitherautomatically or with user input, and their time traces are thencombined to compute a more precise drift correction. In a fourth stage,localizations are pooled from template-based drift markers displayingspots in a defined and spatially resolvable geometry (e.g., 4×3 gridpoints). In a fifth stage, a smoothing of the drift correction isperformed to further reduce noise and enabling the resolution of thefinal image to approach molecular-scale resolution.

Any number and/or combination of these five stages may be performed inaccordance with the techniques described herein. For example, in someembodiments, an amount of drift in the time sequence of images may becharacterized using a quality measure, and based, at least in part, onthe quality measure, one or more of the stages may be eliminated. Inother embodiments, all five stages may be performed, as embodiments arenot limited in this respect. In yet other embodiments, additional driftcorrection stages used in combination with at least one of the stagesdescribed herein may also be used.

In the following description of techniques for performing driftcorrection, the term FWHM (Full Width at Half Maximum) is used as themathematical surrogate for “resolution.” The approximation thatFWHM˜=sigma*2.35 for a Gaussian distribution, where sigma is thestandard deviation, is also used. The techniques described herein forperforming drift correction relate to processing a time sequence ofimages. The recorded image stack is referred to herein as a “movie” andeach of the individual images as a “frame.” Each frame of the movie isoperated on with a spot finding algorithm, and then a local Gaussianfitting algorithm may be used for each identified spot; the spot and itsfitted center position localization are interchangeably referred toherein as a “localization.” The frames of the movie capture one or moretransient events that are present in some frames but not others.

In the illustrative application of the techniques where the frames ofthe movie related to nucleic acid-based imaging as discussed above, thehybridization of an imager strand to a docking strand until theirdisassociation is referred to herein as a binding “event”; thus an eventcould have, and typically will consist of, several localizations in aseries of neighboring frames. Although binding events are discussed infurther detail below as one illustrative transient event that may beanalyzed using the techniques described herein for performing driftcorrection, it should be appreciated that other types of transientevents imaged in a time sequence may alternatively be used. Within acertain area of the field of view, the collection of all localizationsthroughout the entire movie is collectively referred to as the “timetrace,” which reflects the movement of an observed structure, and isused for drift correction in several different ways, as described inmore detail below.

Overview of Drift Correction Techniques

FIG. 12 illustrates a schematic overview of five stages of a driftcorrection procedure that may be performed in accordance with thetechniques described herein. The stages are illustrated as beingperformed consecutively in an order from an unprocessed image to a finalimage that has been processed using the techniques of each of the fivestages. Each of these stages will be discussed in more detail below.Briefly, FIG. 12A(i) illustrates schematics showing the principle ofeach stage of drift correction. In each image, black markers and linesindicate source data, and red values and curves indicate the calculateddrift correction. FIG. 12A(ii) shows a schematic drawing of the majortype of drift markers (e.g., DNA drift markers) used in each stage. FIG.12B(i) illustrates an example structure showing the imaging qualityafter each stage or correction, and FIG. 12B(ii) shows a zoomed image ofthe corresponding green rectangle in FIG. 12B(i) at each stage. Thescale bars shown in FIGS. 12B(i) and 12B(ii) correspond to 50 nm. FIG.12C(i) illustrates an example drift trace after each stage ofcorrection, and FIG. 12C(ii) shows a zoomed image of the correspondinggreen rectangle in FIG. 12C(i) at each stage. The scale bars in FIG.12C(i) correspond to x: 500 nm, t: 500 s, and the scale bars in FIG.12C(ii) correspond to x: 10 nm, t: 10 s. FIG. 18 illustrates analternate representation of stages in a drift correction process inaccordance with some embodiments.

In some embodiments, an image resolution output from the first stage maybe on the order of 1 μm.

In some embodiments, an image resolution output from the second stagemay be on the order of 200 nm.

In some embodiments, an image resolution output from the third stage maybe on the order of 20 nm.

In some embodiments, an image resolution output from the fourth stagemay be on the order of 5 nm.

In some embodiments, an image resolution output from the fourth stagemay be on the order of less than 5 nm.

Imaging Quality and Limit of Achievable Resolution

The finest possible quality of a drift-corrected image is limited by thequality of individual localizations, which is determined by the variousconditions used during an imaging session (e.g., a microscopy imagingsession). To quantitatively assess and effectively compare between thequality of different imaging conditions, a quantity called Distancebetween Neighboring Frame Localizations (DNFL) is defined as the meanseparation between localizations detected from consecutive image frames,which originated from the same transient event (e.g., a binding event).

The procedure for calculating the DNFL for an image is outlined asfollows. For each pair of NF (Neighboring Frames, e.g. frame #1 and #2),all localizations from both frames are pooled and the distance betweenevery pair of localizations from different frames (e.g. one localizationfrom frame #1 versus another from frame #2) is calculated, assuming nodrift between the frames. The resulting distances from all NF pairs arepooled to provide a bimodal distribution. The first mode of the bimodaldistribution is broad, high in amplitude, and spans the width of thefield of view. The second mode of the bimodal distribution is sharp, lowin amplitude, and close to zero, and corresponds to localizations fromthe same binding event. The maximum of the second mode may be determinedand a local Gaussian fitting algorithm may be performed around themaximum to determine the center of the peak. This value may beconsidered the DNFL of a certain image. Without combining localizationsfrom consecutive frames, the DNFL value sets the limit of the finestpossible resolution that can be achieved from a certain image, with amathematical relation between the best achievable resolution and DNFLbeing: best achievable resolution=DNFL/sqrt(2)*2.35.

Drift Correction Quality and Supported Resolution

The quality of a final drift-corrected image may assessed bycharacterizing the Point Spread Function (PSF) of a single binding site.A statistical overlay of images of more than thousands of single dockingsites may be produced as the reference for the PSF distribution. A 2-DGaussian fitting may be performed on the statistical overlay todetermine the standard deviation (sigma) of the PSF, which in turndetermines the best supported resolution of the produced image, given bya similar formula as above: image supported resolution=sigma*2.35. Theisolation and overlay of single isolated docking sites may be performedwith the help of an auxiliary DNA nanostructure. This structure has aknown pattern of well-separated docking sites (e.g., a lattice gridpattern), and may be the same structure used for the template-baseddrift correction stage, discussed in more detail below. Because ofvariation of laser intensity, unevenness of optical surface depositionand other systematic factors, as well as the possible stochastic natureof the imaging process, the above determined image quality of the wholeimage may not reflect the true imaging quality for each single sampleobject in the imaging field. Typically, structures closer to the centerof the imaging field and better fixed to the optical surface, are betterilluminated, and show better resolution than those that are on theperiphery and are less well fixed. The image quality and resolution of asingle molecule may be determined in a procedure similar to the oneabove. A projection of a single molecule of an auxiliary DNAnanostructure (same as above) may be taken along a direction that bestseparates the docking sites (in the case of lattice grid structures,this will be along any of the lattice directions), and a multi-Gaussianfit may be performed on the projected 1-D distribution. The standarddeviation of the fitted Gaussian peaks may then be determined andsimilarly used to infer the resolution of a single-molecule image.

Drift Assessment and Choice of Drift Correction Stages

In some embodiments, the five stages incorporating techniques forperforming drift correction, discussed in further detail below, operatein series to reduce the drift of an unprocessed image, where eachconsecutive stage reduces the drift further, and low enough for thesuccessful operation of the next stage. Depending on the amount of driftin the captured images, less than all five stages may be used. Forexample, if it is determined that there is low drift in the originalimage, the localizations in each frame may be separable, and processingmay begin from the second stage without requiring processing by firststage. If it is determined that there is even lower drift in theoriginal image, processing may be begin from the third stage withoutsignificant loss of final image quality. Due to the complex origin ofdrift, which may involve, among other things, thermal fluctuation andexpansion, microscope stage movement due to electric motor activation,vibration from the building and optical table complex, controlling driftin the original image tends to be difficult, and including the first twostages is often useful in producing a final image with desiredresolution. For example, including all five stages described hereinprovides a robust strategy for drift correction that is applicable toimages taken in most biology labs, without the requirement ofspecialized hardware or building requirements.

In some embodiments, the amount of drift and overall image quality ofthe original unprocessed image may be determined using any suitabletechnique, and the determined image quality may be used to select achoice of drift correction stages to use in performing drift correction.For example, a technique for determining image quality may comparedifferent temporal segments of the same image. In this illustrativetechnique, the original image may be divided into two halves byseparately pooling localizations from the first half and the second halfof the movie, respectively. The cross-correlation between the two imagesmay be calculated and a best offset may be estimated to provide anindication of the overall drift. The indication of overall drift may becompared to one or more threshold values to determine whether one ormore of the drift correction stages may be skipped in performing driftcorrection in accordance with the techniques described herein.

FIG. 13 illustrates a process for performing drift correction inaccordance with some embodiments. In act 210, drift correction isperformed by considering differences in localizations across neighboringframes of a movie. The process then proceeds to act 220, where a singledrift marker is selected, a time trace describing the movement of thedrift marker over time during the movie is determined, and the timetrace for the single drift marker is used to perform drift correction ofthe image. The process then proceeds to act 230, where time traces aredetermined for each of a plurality of drift markers identified in theimage. As discussed in more detail below, differences between the timetraces may be used to provide a further drift correction in the finalimage. The process then proceeds to act 240, where drift correctionusing geometrically-constrained templates is performed. The process thenproceeds to act 250, where the image is further drift corrected bysmoothing the drift trace using suitable smoothing techniques, asdiscussed in more detail below. Each of the five stages for performingdrift correction in accordance with the techniques described herein aredescribed in further detail below. As should be appreciated from theforegoing discussion, not all embodiments require the use of all fivestages for drift correction processing. For example, in someembodiments, only acts 230 and 240 may be performed. In otherembodiments, acts 230, 240, and 250 may be performed. In yet otherembodiments, acts 220, 230, 240 and 250 may be performed withoutincluding act 210.

First Stage Drift Correction

A first stage of drift correction (e.g., act 210 of FIG. 13) operates bycomparing localizations from neighboring frames in a movie. A proceduresimilar to the DNFL calculation described above (or any other suitabletechnique) may be used to identify pairs of localizations originatingfrom the same transient event (e.g., a single binding event). All pairsof localizations originating from the same transient event may be pooledto create a bimodal distribution. After creating a bimodal distributionof the localizations from neighboring images, a cutoff value may beautomatically determined to separate those pairs of localizations fromthe same event (close localizations), from those that are different.Next, all pairs of close localizations for the same neighboring frame(NF) pair are pooled, and the offset between each pair is computed. Thevector average of all offsets are output as the drift correction. For NFpairs with no qualifying close localizations being identified, a zerodrift may be output. The first stage of drift correction typicallycorrects for global drift with high amplitude (farther than 1 μm inoffset), which effectively removes interference between different driftmarkers and allows for incorporation of the next stage. As discussedabove, in some embodiments where different drift markers may already beseparable from each other, the first stage of processing may be omitted.A determination of whether the first stage of processing may be omittedmay be made using an image quality factor analysis, as discussed above,or using any other suitable technique (e.g., manual inspection).

Second Stage Drift Correction

A second stage of drift correction (e.g., act 220 of FIG. 13) operateson a single drift marker or sample object. The single drift marker foruse in this stage of drift correction processing may be selected in anysuitable way. For example, in some embodiments, the single drift markermay be randomly selected from the set of all identified drift markers.In other embodiments, a particular drift marker associated withdesirable qualities (e.g., an average amount of drift over the entiremovie) may be selected as the single drift marker to use for this stage.After selecting the single drift marker, its time trace is automaticallydetermined, smoothed, and output as the drift correction. Alternatively,a drift trace may be manually drawn in cases where separation betweendrift markers is hard to identify automatically.

The second stage of drift correction further reduces global drift in themovie (typically <200 nm), and allows automatic batch identification ofdrift markers in the following stages.

Third Stage of Drift Correction

A third stage of drift correction (e.g., stage 230 of FIG. 2) combinesthe time traces of a plurality of drift markers to compute driftcorrection with a finer resolution than the second stage of driftcorrection. Each drift marker has a large number of docking sites toallow a high temporal coverage; and a large number of these driftmarkers are deposited onto the imaging surface together with thesamples. The number of binding sites on each drift marker and theconcentration of drift markers on the surface may be selectedappropriately to ensure a high quality drift correction, as theimprovement in drift correction in performing this stage is primarilydetermined by the spread of each drift marker in the image and theeffective number of drift markers per frame.

FIG. 14 illustrates a process for performing drift correctioncorresponding to stage 230 of FIG. 2. In act 310, locations of aplurality of drift markers are identified. Identifying locations of theplurality of drift markers may include pooling localizations from allframes of the movie to calculate a two-dimensional (2D) histogram. Then,the locations of drift markers may be identified by appropriately tuningthe histogram binning size and a combination of other selectioncriteria. For example, the binning size of the histogram may be tuned toreflect the feature size of the drift marker, e.g., a fourth or third oftheir overall size. The range of selection criteria includes, but it isnot limited to, a lower-bound threshold of the histogram value andfiltering based on geometrical properties (e.g., area, dimensions).Adequate separation between nearby drift markers is often necessary toexclude false localizations, which, for example, arise from spuriouslocalizations of double-binding events. After the identification of apool (e.g., thousands) of drift markers, the process to act 320, wherethe time trace for each drift marker is determined and the relative timetrace determined as the offset of each time trace from the center of thecombined trace is computed. Because the images capture transient events(e.g., nucleic acid-binding events), not all time traces for a driftmarker may cover all time points in the movie. In such cases, the timetraces may be linearly interpolated at the “missing points” to achieve afiner and smoother result.

After determining the time trace for each of the plurality of driftmarkers, the process proceeds to act 330, where the drift correction forthe image is determined based on the time traces determined for eachdrift marker. Any suitable combination of the time traces may be used todetermine the drift correction, and embodiments are not limited in thisrespect. In some embodiments, a weighted average of the time traces isused to determine the drift correction output from this stage. Forexample, a weighted average of the pool of relative time traces may becomputed as the result of drift correction, where the correction isweighted by the quality of drift marker traces. The quality of driftmarker traces may be determined in any suitable way including, but notlimited to, determining the quality by assessing drift marker quality orindividual localization quality. In some embodiments, the quality ofeach drift marker trace is computed by taking the standard deviation(sigma) of the trace over time. The inverse of this measure (e.g.,1/sigma) for each trace may be used as the weight factor. Alternatively,the quality of each individual localization within the time traces maybe computed as the localization uncertainty given by the formula inThomson, 2002. The inverse of the standard deviation of this calculationmay be used as the weight factor for each time trace. After determiningthe drift correction, the process proceeds to act 340 where the image iscorrected using the determined drift correction.

This stage of drift correction may be performed any number of times. Insome embodiments, this stage of drift correction is iterativelyperformed with different parameters used for each iteration. As driftcorrection proceeds, the remaining drift amplitude is decreased furtherand further, the spatial spread-out of drift markers becomes smaller andsmaller, and selection of drift markers may be performed more and morestringently. Consequently, in initial iterations, the thresholdhistogram count may be set to a lower value, and this value may beadjusted during later iterations to higher values. Additionally, in someembodiments, the valid area and dimensions of drift markers may be setto larger values in initial iterations, and later adjusted to smallervalues during subsequent iterations. Yet further, in some embodiments,separation between drift markers may be shifted from larger values tosmaller values in later iterations. In some embodiments, an interactivequality check (either manually or automatically performed) may bedetermined between iterations to facilitate a determination of furtheroperations.

Depending on the imaging quality, this stage of drift correctiontypically brings the obtained image resolution to within a factor of twofrom the best allowed resolution (i.e. if the precision of eachindividual localization supports resolution of ˜5 nm, then this stageusually yields ˜10 nm resolution). Typically, with good imagingconditions, a resolution <10 nm may be obtained following processingwith this stage.

Fourth Stage of Drift Correction

A fourth stage of drift correction (e.g., act 240 in FIG. 13) uses driftmarker “templates,” and thus this stage is termed “templated driftcorrection.” One or more drift marker templates (e.g., DNAnanostructures with docking sites in a known and well-separatedgeometric arrangement) are deposited onto the imaging surface togetherwith “ordinary” drift markers, discussed above in connection with stagethree. The separation between these docking sites is preferably chosennot to be not smaller than twice the resulting resolution from theprevious stage (e.g., stage three), allowing easy separation betweenlocalizations from different docking sites. The number of docking siteson these templates as well as the concentration of the docking sites onthe surface, is preferably chosen to achieve effective templatecorrection, similar that described for the third stage.

FIG. 15 illustrates a process for performing drift correctioncorresponding to stage 240 of FIG. 2. In act 410, a plurality of driftcorrection templates are identified from a 2-D histogram oflocalizations pooled across all frames of the movie. To distinguish thedrift templates from the drift markers used in the third stage, an extraupper-bound threshold in histogram count may be incorporated in additionto the range of selection criteria as mentioned above. After the drifttemplates have been identified, the process proceeds to act 420, wherethe time trace of each drift template is determined. Because the dockingsites are designed to be well separated in the templates, severalnon-overlapping time traces from individual docking sites may beisolated from each time trace of a full drift template. Thisidentification and separation step may be carried out in a similarmanner as the identification of drift markers from the entire image. Forexample, a combination of local histogram thresholding and filtering onstandard deviation of the individual time traces for each drift templatemay be used.

After the time trace for each drift template has been determined, theprocess proceeds to act 430, where the combination of time traces isused to determine a drift correction for this stage. In someembodiments, this is accomplished by computing relative time traces foreach time trace, and the relative time traces are used, at least inpart, to determine the drift correction. The relative time traces may beused in any suitable way to determine the drift correction. For example,in some embodiments, a weighted average of all the time traces ofindividual docking sites may be averaged to produce the final driftcorrection. The weight factors may be determined in any suitable way.For example, the weight factors may be based on the quality of eachindividual site, or the quality of each individual localization in thetime trace. After determining the drift correction, the process proceedsto act 440 where the image is corrected using the determined driftcorrection.

Depending on the imaging quality, this stage of drift correction mayresult in a resolution of the final image being close to the bestpossible resolution. That is, if the precision of each individuallocalization supports a resolution of ˜5 nm, performing template driftcorrection in accordance with the techniques described herein mayachieve a resolution close to ˜6 nm. In some embodiments, for the <10 nmresolution achieved after the third stage, a DNA nanostructure with 12docking sites arranged in a 4×3 grid of lattice spacing 20 nm may beused as the drift correction template. Template-based drift correctionusing the techniques described in this section may enable theachievement of 6-7 nm resolution after this stage.

Fifth Stage Drift Correction

A fifth stage of drift correction (e.g., act 250 in FIG. 13) performssmoothing of the drift correction trace, and the smoothed driftcorrection trace may be used to perform drift correction. For example,after determining a drift correction for one or more of the second,third and fourth stages discussed above, the resultant drift correctionmay be smoothed using any suitable technique to produce the final driftcorrection result. Smoothing effectively increases the number of driftmarkers or drift templates in each frame, by taking the localizations inneighboring frames into account. Smoothing may be performed in anysuitable manner using any suitable window period. For example, in someembodiments, smoothing is performed with a robust local regressionmethod that operates over a window period determined by thecharacteristic drift time scale. A non-limiting example of a smoothingwindow period may be 10-30 s.

Extension to 3D Imaging

All stages described above can be directly applied to correct a 3D image(e.g., super-resolution image) as well. In 3D super-resolution imaging,for example, an astigmatism lens may be introduced in the imaging pathand the ellipticity of the resulting Gaussian emission profile may beused to determine a z-position of a molecule. The above-describedorigami drift marker structures (e.g., geometric-constrained templates)may be used in a one-to-one fashion to perform the stages of driftcorrection. For the template-based drift correction stage, a DNA origamistructure with a defined 3D shape (e.g., a tetrahedron) may be used.

FIG. 16 illustrates 3D tetrahedrons used as templates for 3D driftcorrection. The four corners are labeled with docking sites. FIG. 16Ashows that the four corners are clearly resolved. FIG. 16B illustratesthe X-Z projection of the structures with a height of ˜85 nm.

Extension to Multicolor Imaging

The above-described techniques for correcting drift in a plurality oftime sequence images is described with respect to imaging a transientevent identified using a single color in the images. However, it shouldbe appreciated that these techniques may be extended to multicolorimaging in which different transient events (e.g., binding of differentnucleic acid drift markers with docking sites) are labeled withdifferent colors that can be identified in the same image. Whenmulticolor imaging is used, the geometric templates discussed above fortemplate-based drift correction may include information describing aparticular known geometry for the different transient events thatcorrespond to the different colors. For example, rather than just asingle binding event occurring at a single docking site in a 3×4 grid,multiple binding events color-coded using different colors in the samegeometric template may be represented, and the drift correction may beperformed using information from the multiple binding events. Otherprocesses for extending the techniques described herein to multicolorimaging are also contemplated, and embodiments are not limited in thisrespect.

Exemplary Computer System

An illustrative implementation of a computer system 600 that may be usedin connection with any of the embodiments of the present disclosuredescribed herein is shown in FIG. 17. The computer system 600 mayinclude one or more processors 610 and one or more computer-readablenon-transitory storage media (e.g., memory 620 and one or morenon-volatile storage media 630). The processor 610 may control writingdata to and reading data from the memory 620 and the non-volatilestorage device 630 in any suitable manner, as the aspects of the presentdisclosure described herein are not limited in this respect. To performany of the functionality described herein, the processor 610 may executeone or more instructions stored in one or more computer-readable storagemedia (e.g., the memory 620), which may serve as non-transitorycomputer-readable storage media storing instructions for execution bythe processor 610.

The above-described embodiments of the present disclosure can beimplemented in any of numerous ways. For example, the embodiments may beimplemented using hardware, software or a combination thereof. Whenimplemented in software, the software code can be executed on anysuitable processor or collection of processors, whether provided in asingle computer or distributed among multiple computers. It should beappreciated that any component or collection of components that performthe functions described above can be generically considered as one ormore controllers that control the above-discussed functions. The one ormore controllers can be implemented in numerous ways, such as withdedicated hardware, or with general purpose hardware (e.g., one or moreprocessors) that is programmed using microcode or software to performthe functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments of the present disclosure comprises at least onenon-transitory computer-readable storage medium (e.g., a computermemory, a floppy disk, a compact disk, a tape, etc.) encoded with acomputer program (i.e., a plurality of instructions), which, whenexecuted on a processor, performs the above-discussed functions of theembodiments of the present disclosure. The computer-readable storagemedium can be transportable such that the program stored thereon can beloaded onto any computer resource to implement the aspects of thepresent disclosure discussed herein. In addition, it should beappreciated that the reference to a computer program which, whenexecuted, performs the above-discussed functions, is not limited to anapplication program running on a host computer. Rather, the termcomputer program is used herein in a generic sense to reference any typeof computer code (e.g., software or microcode) that can be employed toprogram a processor to implement the above-discussed aspects of thepresent disclosure.

Equivalents

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

EXAMPLES Example 1 Cellular Imaging

Multiplexed super-resolution imaging of intra-cellular components infixed cells was achieved by linking docking strands to antibodies (FIG.2). These antibody-DNA conjugates were formed by first reactingbiotinylated docking strands with streptavidin, and then incubating witha biotinylated antibody against the protein of interest. Fixed HeLacells were then immunostained using a preassembled antibody-DNAconjugate against beta-tubulin. Prior to imaging, ATTO655-labeled imagerstrands were introduced to the sample in hybridization buffer (1×PBSsupplemented with 500 mM NaCl), and single-molecule imaging was carriedout using oblique illumination (9). The resulting super-resolutionimages show a clear increase in spatial resolution in contrast to thediffraction-limited representation (FIGS. 3a-3c ). A cross-sectionalprofile taken at position <i> in FIG. 3B yields a distance of ≈79 nmbetween two adjacent microtubules with an apparent width of ≈47 and ≈44nm for each of the microtubules, which is in agreement with earlierreports for immunostained microtubules (13). The antibody-DNAconjugation approach of the present disclosure yields a high labelingdensity, and little to no non-specific binding of imager strands tonon-labeled cellular components occurs.

To demonstrate the multicolor extension of the labeling scheme of thepresent disclosure, where orthogonal imager strand sequences are coupledto spectrally distinct dyes, the microtubule network in a fixed HeLacell was labeled with a preassembled antibody-DNA conjugate carrying adocking sequence for Cy3b-labeled strands, and mitochondria were stainedusing a second antibody linked to an orthogonal sequence for ATTO655imager sequences. While both Cy3b- and ATTO655-labeled imager strandswere present in solution at the same time, imaging was carried outsequentially in the Cy3b and ATTO655 channels and the resultingsuper-resolution images showed a clear increase in spatial resolution ascompared to the diffraction-limited representation (FIGS. 3d-3f ). As inthe single-color case, little-to-no non-specific binding of the imagerstrands to non-labeled components in the cellular environment wasobserved. In addition, similar to the in vitro case, no crosstalkbetween the two colors was observed, indicating a sequence-specificinteraction of the imager strands.

Example 2 Multiplexing

Assuming fixed localization accuracy, one can trivially obtain a directtwo-fold increase in imaging resolution. This can be realized by spacingimaging spots with the same docking strand sequence (e.g., docking sitesa in FIG. 4) farther apart than the actual current resolution limit,thus clearly identifying these sites as single spots in thesuper-resolved image. As all obtained localizations can now be assignedto a specific site, the obtainable imaging resolution is no longer thefull width at half maximum (FWHM) of the reconstructed spots, but ratherthe standard deviation (≈2.35-times smaller than the FWHM). This isdepicted in FIG. 4B1, wherein a set of seven points with 10 nm spacingwas imaged with ≈14 nm resolution, leaving individual pointsunresolvable. Cross-sectional histogram data showed a broad peak(bottom). In FIGS. 4B2 and 4B3, imaging every other site at a timepermitted the localization of individual spots. These localizations werethen combined to form the final composite image with increasedresolution.

FIG. 5A shows a single DNA nanostructure, displaying four distinct setsof DNA sequences, designed to resemble the digits from 0 to 3,respectively. Imaging was performed sequentially using a simple flowchamber setup, first flushing in imager strands complementary to thedocking strands of the number 0, and then exchanging the solution forimager strands with a sequence complementary to docking strands of thenumber 1 and so forth. The resulting images were pseudo-colored torepresent the respective imaging rounds. The demonstration using DNAorigami structures showed the high imaging efficiency as well as nocrosstalk between consecutive imaging runs.

To demonstrate ten-“color” super-resolution imaging of DNA structuresusing Exchange-PAINT, ten unique rectangular DNA origami structures weredesigned, each displaying a distinct pattern of orthogonal dockingstrands that resembles a digit between 0 and 9 (see FIG. 5B(ii) forpattern “4”). After surface immobilization of all ten structures,sequential imaging was performed using a custom made fluidic chamber(FIG. 23A) for easy liquid handling. Ten orthogonal imager strands (P1*to P10*), all labeled with Cy3b, were used to perform Exchange-PAINT.The resulting digits from all ten imaging rounds are shown in FIG.5B(v). Each target is resolved with high spatial resolution.Cross-sectional histograms along the bars of the digits show sub-10 nmFWHM of the distributions (data not shown). Note that high resolution ismaintained for all digits, as the same optimized dye (Cy3b) and imagingconditions are used in each cycle.

FIG. 5B(iii) shows a combined image of all ten rounds, demonstratingspecific interaction of imager strands with respective targets with noobservable crosstalk between cycles. Digits 8 and 9 are not present inthe selected area. An apparent “green” digit 5 instead of 2 was observed(<i> in FIG. 5B(iii)). This is likely not a falsely imaged digit 5 fromcrosstalk, but rather a “mirrored” digit 2. A mirrored image likelyresults from an origami immobilized upside-down, with docking strandstrapped underneath, yet still accessible to imager strands.

The fluidic setup is designed to minimize sample movement by“decoupling” the fluid reservoir and syringe from the actual flowchamber via flexible tubing. To avoid sample distortion, special carewas taken to ensure gentle fluid flow during washing steps. To verifythat the sample indeed exhibited little movement and little-to-nodistortion, a ten-round Exchange-PAINT experiment was performed. The DNAorigami was imaged for digit 4 in the first round and reimaged after tenrounds of buffer exchange. The total sample movement (physical movementof the fluidic chamber with respect to the objective) was less than 2μm, which could easily be corrected using fiducial markers. Normalizedcross-correlation analysis for select structures produced a correlationcoefficient 0.92, demonstrating almost no sample distortion (also seethe discussion in the cellular imaging section).

Finally, using Exchange-PAINT, four different digit patterns weresuccessfully imaged on the same DNA origami structure (FIG. 5B(iv)).Thus Exchange-PAINT is not limited to spatially separate species and canresolve sub-diffraction patterns on the same structure with noobservable crosstalk or sample distortion. Aligning images fromdifferent Exchange-PAINT rounds is straightforward using DNAorigami-based drift markers. Additionally, because imaging is performedusing the same dye, no chromatic aberration needs to be correctedbetween imaging rounds.

The applicability of the methods of the present disclosure in a cellularenvironment was shown by targeting microtubules and mitochondria infixed HeLa cells, similar to FIG. 3, but only using a single colorfluorophore, or spectrally indistinct imager strands. Imaging wasperformed sequentially using imager strands labeled with the same dye(two rounds, FIG. 6).

Example 3 Quantitative Imaging

To demonstrate the feasibility of the quantitative methods of thepresent disclosure, a DNA origami nanostructure with 13 binding sites ina grid-like arrangement was used (FIG. 7C). The incorporation efficiencyfor docking sites was not 100%, leading to a distribution of actuallyincorporated sites (FIGS. 7C and 7D1). Nonetheless, the structures werean ideal test system because the number of available sites could bedetermined visually by counting the number of spots (“direct”) andcomparing it with the corresponding number of sites calculated using theproposed binding kinetic analysis (“kinetics”). FIG. 7D1 shows thebinding site distribution for 377 origami structures obtained by directcounting. The binding site distribution for the same structures obtainedby binding kinetic analysis is shown in FIG. 7D2. Finally, as abenchmark, the “offset” between direct and kinetic counting wascalculated for each structure. The counting “error” or uncertainty forthe method was less than 7% (determined by the coefficient of variationof the Gaussian distribution) with an imaging time of ˜25 min.

Example 4 Kinetic Barcoding

Highly multiplexed super-resolution barcoding was obtained by bindingfrequency analysis rather than geometrical or spectral encoding. Bindingfrequencies of BP-NA conjugates, or docking strands, to a molecule ofinterest is linearly dependent on the number of binding sites on thismolecule. Given a certain concentration of fluorescently-labeled imagerstrands and association rate, binding frequency scales linearly with thenumber of binding sites, and can thus be used for identification. Forexample, 124 distinct dynamic “blinking signatures” were created using 3colors and 4 levels of binding frequency per color (FIG. 8). Compared togeometrical encoding, this approach features much more compact,unstructured probes. Compared to spectral encoding (14) the method ofthe present disclosure is more cost effective, scalable, and easier toimplement. Only 3 fluorescently-labeled imager strands are required inthis frequency encoding method, making it very cost-efficient forhigh-throughput screening experiments. In vitro tests on a DNA origamitest structure are shown in FIG. 8C.

In addition to using the inter-event lifetime τ_(d) or binding frequencyto determine the number of available binding sites and to barcodemolecules, the fluorescence ON-time or τ_(b) and thus the dissociationconstant k_(off) can also be used to encode information. k_(off) can beprecisely tuned by the base-composition and/or length of the duplex ofdocking and imager strand. The feasibility of this approach isillustrated in FIG. 9: 1/τ_(d) and 1/τ_(b) are plotted vs. the length ofthe docking/imaging duplex. 1/τ_(d), and thus k_(on), is independent ofthe duplex stability. However, extending the imaging/docking duplex from9 to 10 nt by adding a single CG base pair reduces the kinetic OFF-rateby almost one order of magnitude.

Finally, a “microbarcode,” a minimal barcode that uses our ability todetect differences in thermodynamic stability of the imager/dockingduplex was produced to identify a large number of molecules using ashort, economical and unstructured probe. The barcode contains only of asingle DNA molecule, roughly 50 nt in length, which is used to tag amolecule of interest using a 21 nt target detection domain t* (FIG. 10A)followed by a ˜30 nt long “barcode” region with a combination of 8, 9,or 10 nt long binding domain for red, green, or blue imager strands.Despite being only 30 nt in length, it can be used to present 3³=27different barcodes with only three spectrally distinct colors and threethermodynamically distinct sequence lengths. FIG. 10 illustrates a8, 9,or 10 nt long docking strands for three colors with a k_(off) of 10, 1and 0.1 per second, respectively. FIG. 10A shows an example barcodeconsisting of an 8 nt long binding domain for a red imager strand, two 9nt long binding domains for the green, and blue imager strand,respectively. This produces characteristic intensity vs. time traceswith increased fluorescence ON-times τ_(b) for the 9 nt interactiondomain compared to the 8 nt interaction domain (FIG. 10B). Stochasticsimulations show that it is clearly possible to distinguish betweenk_(off) values of 10, 1 and 0.1 per second, respectively (FIG. 10C).

Example 5 Genetically Encoded Live-Cell Super-Resolution Imaging

A significant advantage of fluorescence imaging lies in its potential tovisualize biomolecular processes in living cells. There are howeverchallenges in demonstrating live cell super-resolution imaging (ref.22-22). One such challenge is in the delivery of a sufficientconcentration of synthetic imaging probes to living cells in abiocompatible manner while also allowing for proper imaging conditions.Another challenge is the extent of background fluorescence for unboundprobes in the context of a live cell environment where macromolecularcrowding may be more of a significant factor compared to fixed cellenvironments, and “live” helicases may influence binding kinetics.

This disclosure addresses these and other challenges by utilizing agenetically encoded RNA probe that can specifically bind to a targetmolecule in a living cell, and only then start to fluoresce and blink toenable super-resolution imaging of the specific target.

This conditional “blinking” probe is a small single-stranded RNA (<100nt) with a target binding domain (TBD) and a conditional blinking domain(CBD). The CBD is under mechanical control of the TBD and is dark whenthe TBD is not bound to a target T. The binding of TBD with target “T”results in a reconfiguration of the CBD and enables it to blink with anintensity and frequency suitable for the super-resolution imaging of T.

The blinking RNA probe is based on the Spinach aptamer system (ref. 19),a fluorescent RNA mimic of GFP (FIG. 19). Spinach can turn on thefluorescence of an initially dark small molecule DFHBI (similar toDMHBI), which is non-toxic and cell-permeable. By tagging Spinach to atarget RNA, the expression of the target RNA in bacteria and mammaliancells can be imaged (ref. 19).

As the small molecule DFHBI binds and unbinds Spinach, it will alternatebetween a fluorescence ON- and OFF-state. The resulting blinkingbehavior is used to perform super-resolution fluorescence microscopy,which is referred to herein as “Spinach-PAINT” (FIG. 20). Unlike mostlive cell super-resolution imaging techniques, Spinach-PAINT needs nospecial imaging conditions such as special buffer systems or externalphotoswitching or activation. More importantly, DFHBI is a small,cell-permeable molecule and has been shown to provide non-toxic imagingfor living cells. The necessary “blinking” behavior for super-resolutionimaging can be obtained by altering the fluorescence ON- and OFF-timesby adjusting the DFHBI concentration in solution and modifying/mutatingSpinach to enhance/weaken the binding of DFHBI to it in the same spiritas one would tune the DNA-DNA binding interaction of an imaging strandwith its partner. It is possible to characterize and optimize thebinding kinetics of DFHBI to surface-immobilized Spinach based onsingle-molecule measurements (FIG. 20). The binding kinetics are checkedfor compatibility with super-resolution microscopy based on transientbinding.

Generate spinach variants with optimized blinking properties forsuper-resolution imaging: Based on DNA-PAINT, a starting point is toobtain Spinach variants that show ON-times of at least 50 ms, occurringat a rate of ≈2 Hz. It has been found that Spinach exhibits a k_(off) of0.02 s⁻¹, resulting in a residence time of ≈50 s. This is markedlylonger than needed for super-resolution imaging. To identify Spinachvariants with a k_(off) between 1-20 s⁻¹, a “doped” library of Spinachvariants will be prepared, using a dNTP mixture containing the dNTP thatis found in Spinach for each position and the other three dNTPs in a2:1:1:1 ratio. This approach is typically used to optimize aptamersequences (SELEX) (ref. 23). The Spinach variants will then be screenedfor variants with shorter DFHBI residence times. After 5-10 rounds ofSELEX, clones will be individually characterized. A first step is toconfirm that all the Spinach variants still exhibit Spinach-likefluorescence with comparable quantum yield and extinction coefficientupon binding DFHBI. It is generally easy to weaken rather than tostrengthen the binding interaction between an aptamer and a smallmolecule.

A second step involves measuring the binding and unbinding rateconstants of these Spinach variants by single-molecule imaging. Spinachvariants that exhibit binding durations that provide sufficient photoncounts for obtaining precise super-resolution imaging will be chosen.

To optimize the blinking frequency, we will titrate with DFHBI, as therate of RNA-fluorophore complex formation is determined by both k_(on)and the DFHBI concentration, and will identify the concentration thatproduces a blinking rate of 5-10 Hz. Finally, we will have identified aset of Spinach variants that exhibit optimized blinking needed forsuper-resolution imaging.

As a testing platform, we will optimize the super-resolution ability ofSpinach-PAINT by in vitro “imaging” of a DNA-based nanostructure (e.g.,a nanostructure made by DNA origami). The DNA origami substrate has theadvantage of providing a programmable environment for placing multipleSpinach molecules in a defined distance and geometry. This nanoscopicruler system will enable us to precisely quantify the obtainableresolution of this super-resolution imaging technique (FIG. 21).

RNAs that Exhibit Blinking upon Binding Tubulin for Super-ResolutionImaging of Cytoskeleton Proteins: Spinach-based sensors that areactivated by metabolites (ref. 24) and by proteins (ref. 25) have beengenerated. The approach of this disclosure will be used to generateSpinach-based sensors that are activated by binding tubulin, usingtubulin-binding aptamers and the blinking Spinach variants describedherein.

The allosteric form of Spinach is compromised of the modified Spinachaptamer domain fused to a “control” or “sensing” module consisting of anaptamer that binds a target of interest (ref. 24). Binding of the targetresults in the allosteric folding of the modified Spinach aptamer intoan “active” conformation, enabling DFHBI binding and fluorescence (FIG.22). Several tubulin-binding DNA aptamers have been described (ref. 26).This disclosure provides for the evolution of control modules forSpinach capable of sensing tubulin using previously establishedprotocols. Briefly, candidate aptamers will be fused to Spinach toobtain tubulin-dependent Spinach fluorescence, and Spinach activationonly in the presence of tubulin (which will be Cy5 labeled andpolymerized in vitro) will be verified.

Using the conditional Spinach probe generated above and based on thesuper-resolution conditions tested for in vitro imaging, Spinach-PAINTcan be used for, for example, super-resolution imaging of microtubulesin live mammalian cells.

Example 6 Flow Chamber

FIG. 23A shows an example of an experimental setup used for in vitro DNAorigami experiments, as provided herein. The sample is immobilized on aglass coverslip in a PDMS channel. Imaging and washing buffers are addedto a reservoir and pulled through the channel by a syringe. Reservoirsand syringes are connected to the PDMS channel via flexible tubing andare, thus, mechanically decoupled. FIG. 23B shows an experimental setupused for in situ cell imaging. Cells are imaged in a Lab-Tek II chamber.One syringe supplies new buffer solution, the second one removes theprevious buffer.

An exemplary protocol for Exchange-PAINT imaging using a flow chamber,for example, as depicted in FIGS. 23A and 23B, is as follow:

PDMS flow chamber volume: 40 μl

Rinse flow chamber with 100 μl 1 M KOH

Rinse flow chamber with 100 μl buffer A twice

Incubate for 5 min

Rinse flow chamber with 100 μl buffer A

Rinse flow chamber with 50 μl 1 mg/ml BSA-Biotin in buffer A

Incubate for 2 min

Rinse flow chamber with 50 μl 1 mg/ml BSA-Biotin in buffer A

Incubate for 2 min

Rinse flow chamber with 100 μl buffer A twice

Rinse flow chamber with 50 μl 0.5 mg/ml Streptavidin in buffer A

Incubate for 2 min

Rinse flow chamber with 50 μl 0.5 mg/ml Streptavidin in buffer A

Incubate for 2 min

Rinse flow chamber with 100 μl buffer A twice

Rinse flow chamber with 100 μl buffer B twice

Incubate for 30 min

Rinse flow chamber with 100 μl buffer B twice

Rinse flow chamber with 50 μl 1 nM origami in buffer B

Incubate for 10 min

Rinse flow chamber with 100 μl buffer B twice

Attach tubing

Operate in buffer B.

Additional Materials and Methods

Materials. Unmodified DNA oligonucleotides were purchased fromIntegrated DNA Technologies. Fluorescently modified DNA oligonucleotideswere purchased from Biosynthesis. Biotinylated monoclonal antibodiesagainst β-tubulin (9F3; Catalog number: 6181) and COX IV (3E11; Catalognumber: 6014) were purchased from Cell Signaling. Anti-PMP70 (Catalognumber: ab28499) was purchased from Abcam. Anti-TGN46 (Catalog number:NBP1-49643B) was purchased from VWR. Streptavidin was purchased fromInvitrogen (Catalog number: S-888). Bovine serum albumin (BSA), andBSA-biotin was obtained from Sigma Aldrich (Catalog Number: A8549).Glass slides and coverslips were purchased from VWR. Lab-Tek IIchambered cover glass were purchased from Thermo Fisher Scientific.M13mp18 scaffold was obtained from New England Biolabs. p8064 scaffoldfor microtubule-like DNA origami structures was prepared as described19. ‘Freeze N Squeeze’ columns were ordered from Bio-Rad. TetraSpeckBeads were purchased from Life Technologies. Paraformaldehyde,glutaraldehyde and TEM grids (FORMVAR 400 Mesh Copper Grids) wereobtained from Electron Microscopy Sciences. Three buffers were used forsample preparation and imaging: Buffer A (10 mM Tris-HCl, 100 mM NaCl,0.05% Tween-20, pH 7.5), buffer B (5 mM Tris-HCl, 10 mM MgCl2, 1 mMEDTA, 0.05% Tween-20, pH 8), and buffer C (1×PBS, 500 mM NaCl, pH 8).

Optical setup. Fluorescence imaging was carried out on an inverted NikonEclipse Ti microscope (Nikon Instruments) with the Perfect Focus System,applying an objective-type TIRF configuration using a Nikon TIRFilluminator with an oil-immersion objective (CFI Apo TIRF 100×, NA 1.49,Oil). For 2D imaging an additional 1.5 magnification was used to obtaina final magnification of ≈150-fold, corresponding to a pixel size of 107nm. Three lasers were used for excitation: 488 nm (200 mW nominal,Coherent Sapphire), 561 nm (200 mW nominal, Coherent Sapphire) and 647nm (300 mW nominal, MBP Communications). The laser beam was passedthrough cleanup filters (ZT488/10, ZET561/10, and ZET640/20, ChromaTechnology) and coupled into the microscope objective using a multi-bandbeam splitter (ZT488rdc/ZT561rdc/ZT640rdc, Chroma Technology).Fluorescence light was spectrally filtered with emission filters(ET525/50m, ET600/50m, and ET700/75m, Chroma Technology) and imaged onan EMCCD camera (iXon X3 DU-897, Andor Technologies).

DNA origami self-assembly. The microtubule-like DNA origami structureswere formed in a one-pot reaction with 40 μl total volume containing 10nM scaffold strand (p8064), 500 nM folding staples and biotin handles,750 nM biotin anti-handles and 1.1 μM DNA-PAINT docking strands infolding buffer (1× TAE Buffer with 20 mM MgCl₂). The solution wasannealed using a thermal ramp13 cooling from 80° C. to 14° C. over thecourse of 15 h. After self-assembly, monomeric structures were purifiedby agarose gel electrophoresis (1.5% agarose, 0.5× TBE, 10 mM MgCl₂, 1×SybrSafe) at 4.5 V/cm for 1.5 h. Gel bands were cut, crushed and filledinto a ‘Freeze ‘N Squeeze’ column and spun for 5 min at 1000×g at 4° C.Polymerization was carried out at 30° C. for 48 h with a 5-fold excessof polymerization staples in folding buffer. Polymerized structures wereused for imaging without further purification. DNA origami drift markerswere self-assembled in a one-pot reaction (40 μl total volume, 20 nMM13mp18 scaffold, 100 nM biotinylated staples, 530 nM staples withDNA-PAINT docking sites, 1× TAE with 12.5 mM MgCl2). Self-assembledstructures were purified as described before. DNA origami structures forthe 4-“color” in vitro Exchange-PAINT demonstration were self-assembledin a one-pot reaction (40 μl total volume, 30 nM M13mp18 scaffold, 470nM biotinylated staples, 400 nM staples with docking sites for numberimaging, 370 nM core structure staples, 1× TAE with 12.5 mM MgCl2).Self-assembled structures were purified as described before. DNA origamistructures for the 10-“color” in vitro Exchange-PAINT demonstration wereself-assembled in a one-pot reaction (40 μl total volume, 30 nM M13mp18scaffold, 36 nM biotinylated staples, 750 nM staples with docking sitesfor number imaging, 300 nM core structure staples, 1× TAE with 12.5 mMMgCl2). Structures were not purified. Excessive staples are washed outof the sample after immobilization of the structure on the surface. DNAstrand sequences for the microtubule-like DNA origami structures can befound in Table 1. DNA strand sequences for DNA origami drift markers canbe found in Table 2. DNA strand sequences for DNA origami structures for10-“color” in vitro Exchange-PAINT demonstration can be found in Tables3 and 4 for odd and even digits, respectively. DNA strand sequences forDNA origami structures for in vitro Exchange-PAINT demonstration (digits0 to 3) can be found in Table 5. The scaffold sequence for p8064 andM13mp18 correspond to SEQ ID NO: 882 and 883, respectively. DNA-PAINTimager and docking sequences as well as sequences for surface attachmentvia Biotin are listed in Table 6.

Antibody-DNA conjugates. Antibody-DNA conjugates used to specificallylabel proteins of interest with DNA-PAINT docking sites werepreassembled in two steps: First, 3.2 μl of 1 mg/ml streptavidin(dissolved in buffer A) was reacted with 0.5 μl biotinylated DNA-PAINTdocking strands at 100 μM and an additional 5.3 μl of buffer A for 30min at room temperature (RT) while gently shaking. The solution was thenincubated in a second step with 1 μl of monoclonal biotinylatedantibodies at 1 mg/ml against the protein of interest for 30 min at RT.Filter columns (Amicon 100 kDa, Millipore) were used to purify thepreassembled conjugates from unreacted streptavidin-oligo conjugates.

Cell immunostaining. HeLa and DLD1 cells were cultured with Eagle'sminimum essential medium fortified with 10% FBS with penicillin andstreptomycin and were incubated at 37° C. with 5% CO2. Approximately 30%confluence cells per well were seeded into Lab-Tek II chambered coverglass 24 h before fixation. Microtubules, mitochondria, Golgi complex,and peroxisomes were immunostained using the following procedure:washing in PBS; fixation in a mixture of 3% paraformaldehyde and 0.1%glutaraldehyde in PBS for 10 min; 3-times washing with PBS; reductionwith ≈1 mg/ml NaBH4 for 7 min; 3-times washing with PBS;permeabilization with 0.25% (v/v) Triton X-100 in PBS for 10 min;3-times washing with PBS; blocking with 3% (w/v) bovine serum albuminfor 30 min and staining over night with the preassembled antibody-DNAconjugates against β-tubulin, COX IV, PMP70, or TGN46 (conjugates werediluted to 10 μg/ml in 5% BSA); 3-times washing with PBS; post-fixationin a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in PBS for10 min; and 3-times washing with PBS.

Super-resolution DNA-PAINT imaging of microtubule-like DNA origamistructures. For sample preparation, a piece of coverslip (No. 1.5, 18'18mm2, ≈0.17 mm thick) and a glass slide (3×1 inch2, 1 mm thick) weresandwiched together by two strips of double-sided tape to form a flowchamber with inner volume of ≈20 μl. First, 20 μl of biotin-labeledbovine albumin (1 mg/ml, dissolved in buffer A) was flown into thechamber and incubated for 2 min. The chamber was then washed using 40 μlof buffer A. 20 μl of streptavidin (0.5 mg/ml, dissolved in buffer A)was then flown through the chamber and allowed to bind for 2 min. Afterwashing with 40 μl of buffer A and subsequently with 40 μl of buffer B,20 μl of biotin-labeled microtubule-like DNA structures (≈300 pM monomerconcentration) and DNA origami drift markers (≈100 pM) in buffer B werefinally flown into the chamber and incubated for 5 min. The chamber waswashed using 40 μl of buffer B. The final imaging buffer solutioncontained 1.5 nM Cy3b-labeled imager strands in buffer B. The chamberwas sealed with epoxy before subsequent imaging. The CCD readoutbandwidth was set to 1 MHz at 16 bit and 5.1 pre-amp gain. No EM gainwas used. Imaging was performed using TIR illumination with anexcitation intensity of 294 W/cm2 at 561 nm.

Super-resolution Exchange-PAINT imaging of DNA nanostructures. For fluidexchange, a custom flow chamber was constructed. A detailed preparationprotocol can be found in Example 6. Prior to functionalizing the imagingchannel with BSA-biotin, it was rinsed with 1 M KOH for cleaning.Binding of the origami structures to the surface of the flow chamber wasperformed as described before. Each image acquisition step was followedwith a brief ˜1-2 min washing step consisting of at least three washesusing 200 μl of buffer B each. Then the next imager strand solution wasintroduced. The surface was monitored throughout the washing procedureto ensure complete exchange of imager solutions. Acquisition and washingsteps were repeated until all ten targets were imaged. The CCD readoutbandwidth was set to 3 MHz at 14 bit and 5.1 pre-amp gain. No EM gainwas used. Imaging was performed using TIR illumination with anexcitation intensity of 166 W/cm2 at 561 nm (Ten-“color” Exchange-PAINTwith 3 nM Cy3b-labeled imager strands in buffer B, FIG. 5B(iii) and5B(v)) and 600 W/cm2 at 647 nm (Four-“color” Exchange-PAINT with 3 nMATTO655-labeled imager strands in buffer B, FIG. 5B(iv)).

Super-resolution DNA-PAINT imaging of cells. All data was acquired withan EMCCD readout bandwidth of 5 MHz at 14 bit, 5.1 pre-amp gain and 255electron-multiplying gain. Imaging was performed using HILOillumination) 1. The laser power densities were 283 W/cm2 at 647 nm inFIG. 3A, 142 W/cm2 at 647 nm and 19 W/cm2 at 561 nm in FIG. 3D. Imagingconditions: FIG. 3A: 700 pM ATT0655-labeled imager strands in buffer C.FIG.

3D: 600 pM Cy3b-labeled imager strands and 1.5 nM ATT0655-labeled imagerstrands in buffer C.

Super-resolution Exchange-PAINT imaging of cells. A Lab-Tek II chamberwas adapted for fluid exchange. 2D images (FIGS. 6B and 6C) wereacquired with an EMCCD readout bandwidth of 5 MHz at 14 bit, 5.1 preampgain and 255 EM gain. 3D images (FIG. 6) were acquired with a CCDreadout bandwith of 3 MHz at 154 bit, 5.1 pre-amp gain and no EM gain.Imaging was performed using HILO illumination in both cases. Sequentialimaging was done as described for the 2D origami nanostructures, but thewashing steps were performed using buffer C.

3D DNA-PAINT imaging. 3D images were acquired with a cylindrical lens inthe detection path (Nikon). The N-STORM analysis package for NISElements (Nikon) was used for data processing. Imaging was performedwithout additional magnification in the detection path, yielding 160 nmpixel size. 3D calibration was carried out according to themanufacturer's instructions.

Imager strand concentration determination. Optimal imager concentrationsare determined empirically according to the labeling density. Generally,a high enough fluorescence OFF/ON-ratio has to be ensured in order toguarantee binding of only a single imager strand per diffraction-limitedarea. Additionally, a sufficient imager strand concentration (and thussufficiently low fluorescence OFF-time) is necessary to ensuresufficient binding events and thereby robust detection of every dockingstrand during image acquisition.

Super-resolution data processing. Super-resolution DNA-PAINT images werereconstructed using spot-finding and 2D-Gaussian fitting algorithmsprogrammed in LabVIEW10. A simplified version of this software isavailable for download at the DNA-Paint website (org suffix).

Normalized cross-correlation analysis. Normalized cross-correlationcoefficients were obtained by first normalizing the respectivereconstructed gray-scale super-resolution images and subsequentlyperforming a cross-correlation analysis in MATLAB R2013b (MathWorks,Natick, Mass., USA).

Drift correction and channel alignment. DNA origami structures are usedfor drift correction and as alignment markers in in vitro DNA-PAINT andExchange-PAINT imaging. Drift correction was performed by tracking theposition of each origami drift marker throughout the duration of eachmovie. The trajectories of all detected drift markers were then averagedand used to globally correct the drift in the final super-resolutionreconstruction. For channel alignment between different imaging cyclesin Exchange-PAINT, these structures are used as alignment points bymatching their positions in each Exchange-PAINT image. For cellularimaging, 100 nm gold nanoparticles (Sigma Aldrich;10 nM in buffer C,added before imaging) were used as drift and alignment markers. The goldnanoparticles adsorb non-specifically to the glass bottom of the imagingchambers. Drift correction and alignment is performed in a similarfashion as for the origami drift markers. Again, the apparent movementof all gold nanoparticles in a field of view is tracked throughout themovie. The obtained trajectories are then averaged and used for globaldrift correction of the final super-resolution image. For the dual-colorimage of mitochondria and microtubules in FIG. 3D-3F, the gold particlesare visible in both color channels. The same gold nanoparticles are alsoused for drift-correction and re-alignment of the different imagingrounds in the in situ Exchange-PAINT experiments (FIG. 6).

Transmission electron microscopy imaging. For imaging, 3.5 μl ofundiluted microtubules-like DNA structures were adsorbed for 2 minutesonto glow-discharged, carbon-coated TEM grids. The grids were thenstained for 10 seconds using a 2% aqueous ultra-filtrated (0.2 μmfilter) uranyl formate solution containing 25 mM NaOH. Imaging wasperformed using a JEOL JEM-1400 operated at 80 kV.

Atomic force microscopy imaging. Imaging was performed using tappingmode on a Multimode VIII atomic force microscope (AFM) with an E-scanner(Bruker). Imaging was performed in TAE/Mg2+ buffer solution with DNP-Soxide-sharpened silicon nitride cantilevers and SNL sharp nitride levers(Bruker Probes) using resonance frequencies between 7-9 kHz of thenarrow 100 μm, 0.38 N/m force constant cantilever. After self-assemblyof the origami structure ≈20 μl of TAE/Mg2+ buffer solution wasdeposited onto a freshly cleaved mica surface (Ted Pella) glued to ametal puck (Ted Pella). After 30 s the mica surface was dried using agentle stream of N2 and 5 μl of the origami solution was deposited ontothe mica surface. After another 30 s, 30 μl of additional buffersolution was added to the sample. Imaging parameters were optimized forbest image quality while maintaining the highest possible setpoint tominimize damage to the samples. Images were post-processed bysubtracting a 1st order polynomial from each scan line. Drive amplitudeswere approximately 0.11 V, integral gains ≈2, and proportional gains ≈4.

In Tables 1-5 below, each oligonucleotide position in a describedstructure is set forth. Each oligonucleotide position (e.g., n[n]n[n])in the first column is separated by a comma and corresponds respectivelyto a sequence identification number in the second column within the samerow. Each sequence identification number identifies a correspondingoligonucleotide sequence in the attached sequence listing, incorporatedby reference herein. For example, in Table 1, position 0[39]21[39]corresponds to the oligonucleotide represented by SEQ ID NO: 1; position0[79]1[79] corresponds to the oligonucleotide represented by SEQ ID NO:2, and so forth.

TABLE 1 Staple sequences for microtubule analog DNA structure. SequencePosition* Identifiers Description 0[39]21[39], 0[79]1[79],0[167]22[168], 0[199]2[200], 0[239]21[231], SEQ ID NO: 1-SEQ Structure1[24]18[24], 1[96]17[95], 1[120]1[151], 1[152]19[167], 2[39]23[55], IDNO: 178 Strand 2[79]23[71], 2[103]3[119], 2[127]31[143], 2[199]23[207],2[231]5[231], 3[16]31[31], 3[56]19[55], 3[80]2[80], 3[120]24[128],3[168]5[175], 4[71]5[87], 4[135]22[120], 4[207]6[184], 5[16]22[16],5[32]25[31], 5[52]3[55], 5[88]23[103], 5[152]4[136], 5[176]22[184],6[95]4[72], 6[127]8[120], 6[151]22[144], 6[159]26[160], 6[183]2[184],6[207]4[208], 6[231]24[208], 7[16]4[16], 7[48]5[51], 7[80]25[95],7[112]6[96], 7[176]25[191], 8[39]11[31], 8[95]12[80], 8[159]10[160],8[191]8[160], 9[48]25[63], 9[104]24[112], 9[120]10[136], 9[136]24[144],9[176]11[191], 9[208]25[223], 10[31]27[23], 10[55]8[40], 10[95]27[95],10[135]26[112], 10[159]27[159], 10[183]27[191], 10[215]10[184],11[192]26[208], 12[31]29[23], 12[55]28[32], 12[79]11[63],12[119]16[120], 12[183]29[191], 12[215]27[215], 13[40]27[55],13[72]30[80], 13[104]9[103], 13[144]17[159], 13[168]25[183],13[216]14[224], 14[55]16[40], 14[87]28[72], 14[119]15[135],14[223]30[208], 15[32]29[55], 15[80]18[80], 15[96]28[104],15[160]28[168], 16[119]31[111], 16[143]30[120], 16[191]14[160],16[207]19[207] 16[239]29[231], 17[24]14[24], 17[40]18[56], 17[64]14[56],17[160]31[175], 17[224]31[239], 18[55]2[40], 18[79]21[79], 18[111]0[96],18[183]30[160], 18[239]2[232], 19[56]30[64], 19[96]30[96],19[168]3[167], 19[192]30[184], 19[208]30[224], 20[159]21[135],20[223]21[199], 21[16]1[23], 21[40]4[32], 21[80]22[96], 21[136]2[128],21[200]1[215], 21[232]6[232], 22[95]3[79], 22[119]2[104],22[143]21[159], 22[167]7[175], 22[183]18[184], 22[207]21[223],23[56]1[55], 23[72]24[56], 23[104]5[103], 23[208]22[208], 24[79]7[79],24[127]9[135], 24[143]5[151], 24[231]26[216], 25[64]24[80],25[224]10[216], 26[159]12[144], 26[207]6[208], 26[239]7[247],27[96]10[96] 27[112]14[120], 27[136]7[151], 28[191]15[183],29[56]15[79], 29[136]27[135], 29[192]13[215], 29[232]28[216],30[95]14[88], 30[119]29[135], 30[183]16[192], 30[207]17[223],30[223]15[239], 31[112]1[119], 31[144]19[143], 1[56]0[40], 1[80]19[95],1[216]0[200], 2[183]19[191], 5[104]6[128], 8[63]10[56], 9[80]8[64],11[64]9[79], 11[216]9[239], 14[159]15[159], 15[184]16[208],17[96]15[95], 19[128]18[112], 19[144]19[127], 24[55]7[47],24[111]7[111], 24[183]6[160], 24[207]8[192], 25[32]9[47], 25[96]8[96],25[192]9[207], 27[32]10[32], 27[160]9[175], 30[63]17[63], 30[79]0[80],30[159]16[144], 31[32]15[31], 31[176]0[168], 13[136]12[120],15[136]13[135] 27[24]13[39], 27[56]12[56], 27[216]12[216], 28[71]13[71],28[103]13[103], 28[167]13[167], 28[215]12[184] 1[56]0[40], 1[80]19[95],1[216]0[200], 2[183]19[191], 5[104]6[128], SEQ ID NO: 179-SEQ DNA-PAINT,8[63]10[56], 9[80]8[64], 11[64]9[79], 11[216]9[239], 14[159]15[159], IDNO: 206 docking site 15[184]16[208], 17[96]15[95], 19[128]18[112],19[144]19[127], 24[55]7[47], 24[111]7[111], 24[183]6[160],24[207]8[192], 25[32]9[47], 25[96]8[96], 25[192]9[207], 27[32]10[32],27[160]9[175], 30[63]17[63], 30[79]0[80], 30[159]16[144], 31[32]15[31],31[176]0[168] 2[9]2[10], 4[15]6[248], 5[232]5[15], 6[247]3[15],7[248]24[232], 9[240]11[7], SEQ ID NO: 207-SEQ Connector 11[8]25[23],13[8]26[240], 14[23]16[240], 15[240]28[8], 17[8]18[240], ID NO: 225strand 19[3]0[248], 22[15]21[15], 24[23]7[15], 27[237]13[7],28[7]27[236], 30[23]17[7], 31[16]31[15], 31[240]0[240] 13[136]12[120],15[136]13[135], 27[24]13[39], 27[56]12[56], 27[216]12[216], SEQ ID NO:226-SEQ 3′-Biotin, 28[71] 13[71], 28[103] 13[103], 28[167] 13[167],28[215] 12[184] ID NO: 234 docking site

TABLE 2 Staple sequences for drift markers. Sequence Position*Identifiers Description 0[111]1[95], 0[143]1[127], 0[175]0[144],0[207]1[191], 0[239]1[223], SEQ ID NO: 235-SEQ DNA-PAINT, 1[32]3[31],1[96]3[95], 1[224]3[223], 2[79]0[80], 2[111]0[112], 2[143]1[159], ID NO:402 docking site 2[175]0[176], 2[207]0[208], 3[32]5[31], 3[96]5[95],3[160]4[144], 3[224]5[223], 4[143]3[159], 5[32]7[31], 5[96]7[95],5[224]7[223], 6[47]4[48], 6[79]4[80], 6[111]4[112], 6[143]5[159],6[175]4[176], 6[207]4[208], 6[239]4[240], 6[271]4[272], 7[32]9[31],7[96]9[95], 7[160]8[144], 7[224]9[223], 8[143]7[159], 9[32]11[31],9[64]11[63], 9[96]11[95], 9[128]11[127], 9[192]11[191], 9[224]11[223],9[256]11[255], 10[47]8[48], 10[79]8[80], 10[111]8[112], 10[143]9[159],10[175]8[176], 10[207]8[208], 10[239]8[240], 10[271]8[272],12[143]11[159], 13[32]15[31], 13[64]15[63], 13[96]15[95],13[128]15[127], 13[192]15[191], 13[224]15[223], 13[256]15[255],14[271]12[272], 15[32]17[31], 15[96]17[95], 15[160]16[144],15[224]17[223], 16[143]15[159], 17[32]19[31], 17[96]19[95],17[224]19[223], 18[47]16[48], 18[79]16[80], 18[111]16[112],18[143]17[159], 18[175]16[176], 18[207]16[208], 18[239]16[240],18[271]16[272], 19[32]21[31], 19[96]21[95], 19[160]20[144],19[224]21[223], 20[143]19[159], 21[96]23[95], 21[160]22[144],21[224]23[223], 22[47]20[48], 22[79]20[80], 22[111]20[112],22[143]21[159], 22[175]20[176], 22[207]20[208], 22[239]20[240],22[271]20[272], 23[64]22[80], 23[96]22[112], 23[128]23[159],23[160]22[176], 23[192]22[208], 7[56]9[63], 7[120]9[127], 7[184]9[191],7[248]9[255], 11[32]13[31], 11[64]13[63], 11[96]13[95], 11[128]13[127],11[160]12[144], 11[192]13[191], 11[224]13[223], 11[256]13[255],14[47]12[48], 14[79]12[80], 14[111]12[112], 14[143]13[159],14[175]12[176], 14[207]12[208], 14[239]12[240], 21[120]23[127],21[184]23[191], 1[160]2[144], 4[47]2[48], 4[79]2[80], 4[111]2[112],4[175]2[176], 4[207]2[208], 4[239]2[240], 4[271]2[272], 5[160]6[144],8[47]6[48], 8[79]6[80], 8[111]6[112], 8[175]6[176], 8[207]6[208],8[239]6[240], 8[271]6[272], 9[160]10[144], 12[47]10[48], 12[79]10[80],12[111]10[112], 12[175]10[176], 12[207]10[208], 12[239]10[240],12[271]10[272], 13[160]14[144], 16[47]14[48], 16[79]14[80],16[111]14[112], 16[175]14[176], 16[207]14[208], 16[239]14[240],16[271]14[272], 17[160]18[144], 20[47]18[48], 20[79]18[80],20[111]18[112], 20[175]18[176], 20[207]18[208], 20[239]18[240],20[271]18[272], 0[47]1[31], 0[79]1[63], 0[271]1[255], 2[47]0[48],2[239]0[240], 2[271]0[272], 21[32]23[31], 21[56]23[63], 21[248]23[255],23[32]22[48], 23[224]22[240], 23[256]22[272] 4[63]6[56], 4[127]6[120],4[191]6[184], 4[255]6[248], 18[63]20[56], SEQ ID NO: 403-SEQ 5′-Biotin18[127]20[120], 18[191]20[184], 18[255]20[248] ID NO: 410 modification1[64]4[64], 1[128]4[128], 1[192]4[192], 1[256]4[256], 15[64]18[64], SEQID NO: 411-SEQ Structure 15[128]18[128], 15[192]18[192], 15[256]18[256],ID NO: 418 strand

TABLE 3 Staple sequences for DNA origami structures for 10-“color” invitro Exchange- PAINT demonstration (odd digits). Sequence DescriptionPosition* Identifiers (number) 0[111] 1[95], 0[143]1[127], 0[175]0[144],0[79]1[63], 1[160]2[144], 2[47]0[48], SEQ ID NO: 419-SEQ 5, 93[160]4[144], 5[160]6[144], 7[160]8[144] ID NO: 427 10[271]8[272],11[160]12[144], 12[271]10[272], 13[160]14[144], SEQ ID NO: 428-SEQ 3, 5,9 14[271]12[272], 15[160]16[144], 16[271]14[272], 17[160]18[144], ID NO:445 18[271]16[272], 19[160]20[144], 2[271]0[272], 20[271]18[272],21[160]22[144], 22[271]20[272], 4[271]2[272], 6[271]4[272],8[271]6[272], 9[160]10[144] 0[47]1[31], 1[32]3[31], 11[32]13[31],13[32]15[31], 15[32]17[31], SEQ ID NO: 446-SEQ 3, 5, 7, 9 17[32]19[31],19[32]21[31], 3[32]5[31], 5[32]7[31], 7[32]9[31], 9[32]11[31] ID NO: 45621[120]23[127], 21[56]23[63], 21[96]23[95], 23[32]22[48], 23[64]22[80],SEQ ID NO: 457-SEQ 1, 3, 7, 9 23[96]22[112] ID NO: 462 21[184]23[191],21[224]23[223], 21[248]23[255], 21[32]23[31], SEQ ID NO: 463-SEQ 1, 3,5, 7, 9 23[128]23[159], 23[160]22[176], 23[192]22[208], 23[224]22[240],ID NO: 471 23[256]22[272] 2[111]0[112], 2[79]0[80] SEQ ID NO: 472-SEQ 9ID NO: 473 0[207]1[191], 0[239]1[223], 0[271]1[255], 1[128]4[128],1[192]4[192], SEQ ID NO: 474-SEQ Structure 1[224]3[223], 1[256]4[256],1[64]4[64], 1[96]3[95], 10[111]8[112], ID NO: 594 staple 10[143]9[159],10[175]8[176], 10[207]8[208], 10[239]8[240], 10[47]8[48], 10[79]8[80],11[128]13[127], 11[192]13[191], 11[224]13[223], 11[256]13[255],11[64]13[63], 11[96]13[95], 12[111]10[112], 12[143]11[159],12[175]10[176], 12[207]10[208], 12[239]10[240], 12[47]10[48],12[79]10[80], 13[128]15[127], 13[192]15[191], 13[224]15[223],13[256]15[255], 13[64]15[63], 13[96]15[95], 14[111]12[112],14[143]13[159], 14[175]12[176], 14[207]12[208], 14[239]12[240],14[47]12[48], 14[79]12[80], 15[128]18[128], 15[192]18[192],15[224]17[223], 15[256]18[256], 15[64]18[64], 15[96]17[95],16[111]14[112], 16[143]15[159], 16[175]14[176], 16[207]14[208],16[239]14[240], 16[47]14[48], 16[79]14[80], 17[224]19[223],17[96]19[95], 18[111]16[112], 18[143]17[159], 18[175]16[176],18[207]16[208], 18[239]16[240], 18[47]16[48], 18[79]16[80],19[224]21[223], 19[96]21[95], 2[143]1[159], 2[175]0[176], 2[207]0[208],2[239]0[240], 20[111]18[112], 20[143]19[159], 20[175]18[176],20[207]18[208], 20[239]18[240], 20[47]18[48], 20[79]18[80],22[111]20[112], 22[143]21[159], 22[175]20[176], 22[207]20[208],22[239]20[240], 22[47]20[48], 22[79]20[80], 3[224]5[223], 3[96]5[95],4[111]2[112], 4[143]3[159], 4[175]2[176], 4[207]2[208], 4[239]2[240],4[47]2[48], 4[79]2[80], 5[224]7[223], 5[96]7[95], 6[111]4[112],6[143]5[159], 6[175]4[176], 6[207]4[208], 6[239]4[240], 6[47]4[48],6[79]4[80], 7[120]9[127], 7[184]9[191], 7[224]9[223], 7[248]9[255],7[56]9[63], 7[96]9[95], 8[111]6[112], 8[143]7[159], 8[175]6[176],8[207]6[208], 8[239]6[240], 8[47]6[48], 8[79]6[80], 9[128]11[127],9[192]11[191], 9[224]11[223], 9[256]11[255], 9[64]11[63], 9[96]11[95]4[63]6[56], 4[127]6[120], 4[191]6[184], 4[255]6[248], 18[63]20[56], SEQID NO: 595-SEQ 5′-Biotin 18[127]20[120], 18[191]20[184], 18[255]20[248]ID NO: 602

TABLE 4 Staple sequences for DNA origami structures for 10-“color” invitro Exchange- PAINT demonstration (even digits). Sequence DescriptionPosition* Identifiers (number) 1[160]2[144], 11[160]12[144],13[160]14[144], 15[160]16[144], SEQ ID NO: 603-SEQ 2, 4, 6, 817[160]18[144], 19[160]20[144], 21[160]22[144], 3[160]4[144],5[160]6[144], ID NO: 613 7[160]8[144], 9[160]10[144] 21[224]23[223],21[248]23[255] SEQ ID NO: 614-SEQ 0, 4, 8 ID NO: 615 0[111]1[95],0[143]1[127], 0[79]1[63], 2[111]0[112], 2[47]0[48], 2[79]0[80], SEQ IDNO: 606-SEQ 0, 4, 6, 8 21[184]23[191], 23[160]22[176], 23[192]22[208],23[224]22[240] ID NO: 625 1[32]3[31], 11[32]13[31], 13[32]15[31],15[32]17[31], 17[32]19[31], SEQ ID NO: 626-SEQ 0, 2, 8 19[32]21[31],3[32]5[31], 5[32]7[31], 7[32]9[31], 9[32]11[31] ID NO: 635 0[207]1[191],0[239]1[223], 0[271]1[255], 10[271]8[272], 12[271]10[272], SEQ ID NO:636-SEQ 0, 2, 6, 8 14[271]12[272], 16[271]14[272], 18[271]16[272],2[175]0[176], 2[207]0[208], ID NO: 652 2[239]0[240], 2[271]0[272],20[271]18[272], 22[271]20[272], 4[271]2[272], 6[271]4[272], 8[271]6[272]21[32]23[31], 21[56]23[63], 21[96]23[95], 23[32]22[48], 23[64]22[80],SEQ ID NO: 653-SEQ 0, 2, 4, 8 23[96]22[112] ID NO: 658 0[175]0[144],0[47]1[31], 23[128]23[159], 23[256]22[272] SEQ ID NO: 659-SEQ 0, 2, 4,6, 8 ID NO: 662 21[120]23[127] SEQ ID NO: 663 0, 2, 4 1[128]4[128],1[192]4[192], 1[224]3[223], 1[256]4[256], 1[64]4[64], SEQ ID NO: 664-SEQStructure 1[96]3[95], 10[111]8[112], 10[143]9[159], 10[175]8[176],10[207]8[208], ID NO: 778 Staple 10[239]8[240], 10[47]8[48],10[79]8[80], 11[128]13[127], 11[192]13[191], 11[224]13[223],11[256]13[255], 11[64]13[63], 11[96]13[95], 12[111]10[112],12[143]11[159], 12[175]10[176], 12[207]10[208], 12[239]10[240],12[47]10[48], 12[79]10[80], 13[128]15[127], 13[192]15[191],13[224]15[223], 13[256]15[255], 13[64]15[63], 13[96]15[95],14[111]12[112], 14[143]13[159], 14[175]12[176], 14[207]12[208],14[239]12[240], 14[47]12[48], 14[79]12[80], 15[128]18[128],15[192]18[192], 15[224]17[223], 15[256]18[256], 15[64]18[64],15[96]17[95], 16[111]14[112], 16[143]15[159], 16[175]14[176],16[207]14[208], 16[239]14[240], 16[47]14[48], 16[79]14[80],17[224]19[223], 17[96]19[95], 18[111]16[112], 18[143]17[159],18[175]16[176], 18[207]16[208], 18[239]16[240], 18[47]16[48],18[79]16[80], 19[224]21[223], 19[96]21[95], 2[143]1[159],20[111]18[112], 20[143]19[159], 20[175]18[176], 20[207]18[208],20[239]18[240], 20[47]18[48], 20[79]18[80], 22[111]20[112],22[143]21[159], 22[175]20[176], 22[207]20[208], 22[239]20[240],22[47]20[48], 22[79]20[80], 3[224]5[223], 3[96]5[95], 4[111]2[112],4[143]3[159], 4[175]2[176], 4[207]2[208], 4[239]2[240], 4[47]2[48],4[79]2[80], 5[224]7[223], 5[96]7[95], 6[111]4[112], 6[143]5[159],6[175]4[176], 6[207]4[208], 6[239]4[240], 6[47]4[48], 6[79]4[80],7[120]9[127], 7[184]9[191], 7[224]9[223], 7[248]9[255], 7[56]9[63],7[96]9[95], 8[111]6[112], 8[143]7[159], 8[175]6[176], 8[207]6[208],8[239]6[240], 8[47]6[48], 8[79]6[80], 9[128]11[127], 9[192]11[191],9[224]11[223], 9[256]11[255], 9[64]11[63], 9[96]11[95] 4[63]6[56],4[255]6[248], 4[191]6[184], 4[127]6[120], 18[63]20[56], SEQ ID NO:779-SEQ 5′-Biotin 18[255]20[248], 18[191]20[184], 18[127]20[120], ID NO:786

TABLE 5 Staple sequences for DNA origami structures for in vitroExchange-PAINT demonstration (digits 0 to 3) Sequence DescriptionPosition* Identifiers (number) 2[47]0[48], 2[79]0[80], 2[111]0[112],2[143]1[159], 2[175]0[176], SEQ ID NO: 787-SEQ 0 2[207]0[208],2[239]0[240], 6[47]4[48], 6[239]4[240], 10[47]8[48], ID NO: 80810[239]8[240], 14[47]12[48], 14[239]12[240], 18[47]16[48],18[239]16[240], 22[47]20[48], 22[79]20[80], 22[111]20[112],22[143]21[159], 22[175]20[176], 22[207]20[208], 22[239]20[240]9[64]11[63], 9[96]11[95], 9[128]11[127], 9[192]11[191], 9[224]11[223],SEQ ID NO: 809-SEQ 1 9[256]11[255], 11[64]13[63], 11[96]13[95],11[128]13[127], 11[160]12[144], ID NO: 832 11[192]13[191],11[224]13[223], 11[256]13[255], 12[47]10[48], 12[79]10[80],12[111]10[112], 12[175]10[176], 12[207]10[208], 12[239]10[240],13[160]14[144], 14[79]12[80], 14[111]12[112], 14[175]12[176],14[207]12[208] 0[175]0[144], 0[207]1[191], 0[239]1[223], 0[271]1[255],1[32]3[31], SEQ ID NO: 833-SEQ 2 4[143]3[159], 4[271]2[272], 5[32]7[31],8[143]7[159], 8[271]6[272], ID NO: 857 9[32]11[31], 12[143]11[159],12[271]10[272], 13[32]15[31], 16[143]15[159], 16[271]14[272],17[32]19[31], 20[143]19[159], 20[271]18[272], 21[32]23[31],21[56]23[63], 21[96]23[95], 21[120]23[127], 21[160]22[144],23[256]22[272] 0[47]1[31], 2[271]0[272], 3[32]5[31], 6[143]5[159],6[271]4[272], 7[32]9[31], SEQ ID NO: 858-SEQ 3 10[143]9[159],10[271]8[272], 11[32]13[31], 14[143]13[159], 14[271]12[272], ID NO: 88115[32]17[31], 18[143]17[159], 18[271]16[272], 19[32]21[31],19[160]20[144], 22[271]20[272], 23[32]22[48], 23[64]22[80],23[96]22[112], 23[128]23[159], 23[160]22[176], 23[192]22[208],23[224]22[240]

TABLE 6DNA-PAINT docking and imager sequences and biotin docking sequenceSEQ ID Description NO: Sequence Imager P1* 884 5′-CTAGATGTATdyeImager P2* 885 5′-TATGTAGATC-dye Imager P3* 886 5′-GTAATGAAGA-dyeImager P4* 887 5′-GTAGATTCAT-dye Imager P5* 888 5′-CTTTACCTAA-dyeImager P6* 889 5′-GTACTCAATT-dye Imager P7* 890 5′-CATCCTAATT-dyeImager P8* 891 5′-GATCCATTAT-dye Imager P9* 892 5′-CACCTTATTA-dyeImager P10* 893 5′-CCTTCTCTAT-dye Imager P11* 894 5′-GTATCATCAA-dyeImager P12* 895 5′-GAATCACTAT-dye 9nt P1 docking site 896Strand-TTATACATCTA-3′ 9nt P2 docking site 897 Strand-TTATCTACATA-3′10nt P2 docking site 898 Strand-TTGATCTACATA-3′ 9nt P3 docking site 899Strand-TTTCTTCATTA-3′ 9nt P4 docking site 900 Strand-TTATGAATCTA-3′9nt P5 docking site 901 Strand-TTTTAGGTAAA-3′ 9nt P6 docking site 902Strand-TTAATTGAGTA-3′ 9nt P7 docking site 903 Strand-TTAATTAGGAT-3′9nt P8 docking site 904 Strand-TTATAATGGAT-3′ 9nt P9 docking site 905Strand-TTTAATAAGGT-3′ 9nt P10 docking site 906 Strand-TTATAGAGAAG-3′9nt P11 docking site 907 Strand-TTTTGATGATA-3′ 9nt P12 docking site 908Strand-TTATAGTGATT-3′ Biotinylated P1 docking  909 Biotin-TTATACATCTA-3′site for antibody coupling Biotinylated P2 docking  910Biotin-TTATCTACATA-3′ site for antibody couplingBiotinylated P3 docking  911 Biotin-TTTCTTCATTA-3′site for antibody coupling Biotinylated P4 docking  912Biotin-TTATGAATCTA-3′ site for antibody couplingBiotinylated docking site for 913 Biotin-GAATCGGTCACAGTACAACCG-3′microtubule-like structure

REFERENCES

-   1. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging    by stochastic optical reconstruction microscopy (STORM). Nat Methods    3, 793-5 (2006).-   2. Hell, S. W. Microscopy and its focal switch. Nature methods 6,    24-32 (2009).-   3. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution    limit by stimulated emission: stimulated-emission-depletion    fluorescence microscopy. Opt Lett 19, 780-2 (1994).-   4. Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W.,    Olenych, S., Bonifacino, J. S., Davidson, M. W.,    Lippincott-Schwartz, J. & Hess, H. F. Imaging intracellular    fluorescent proteins at nanometer resolution. Science 313, 1642-5    (2006).-   5. Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction    imaging by accumulated binding of diffusing probes. Proceedings of    the National Academy of Sciences of the United States of America    103, 18911-18916 (2006).-   6. Giannone, G., Hosy, E., Levet, F., Constals, A., Schulze, K.,    Sobolevsky, A. I., Rosconi, M. P., Gouaux, E., Tampe, R.,    Choquet, D. & Cognet, L. Dynamic superresolution imaging of    endogenous proteins on living cells at ultra-high density. Biophys J    99, 1303-10 (2010).-   7. Lew, M. D., Lee, S. F., Ptacin, J. L., Lee, M. K., Twieg, R. J.,    Shapiro, L. & Moerner, W. E. Three-dimensional superresolution    colocalization of intracellular protein superstructures and the cell    surface in live Caulobacter crescentus. Proc Natl Acad Sci USA 108,    E1102-10 (2011).-   8. Jungmann, R., Steinhauer, C., Scheible, M., Kuzyk, A.,    Tinnefeld, P. & Simmel, F. C. Single-Molecule Kinetics and    Super-Resolution Microscopy by Fluorescence Imaging of Transient    Binding on DNA Origami. Nano Letters 10, 4756-4761 (2010).-   9. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined    thin illumination enables clear single-molecule imaging in cells.    Nature Methods 5, 159-161 (2008).-   10. Lin, C., Jungmann, R., Leifer, A. M., Li, C., Levner, D.,    Church, G. M., Shih, W. M. & Yin, P. Submicrometre geometrically    encoded fluorescent barcodes self-assembled from DNA. Nat Chem 4,    832-9 (2012).-   11. Derr, N. D., Goodman, B. S., Jungmann, R., Leschziner, A. E.,    Shih, W. M. & Reck-Peterson, S. L. Tug-of-war in motor protein    ensembles revealed with a programmable DNA origami scaffold. Science    338, 662-5 (2012).-   12. Johnson-Buck, A., Nangreave, J., Kim, D. N., Bathe, M., Yan, H.    & Walter, N. G. Super-resolution fingerprinting detects chemical    reactions and idiosyncrasies of single DNA pegboards. Nano Lett 13,    728-33 (2013).-   13. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A    simple, versatile method for GFP-based super-resolution microscopy    via nanobodies. Nat Methods 9, 582-4 (2012).-   14. Lubeck, E. & Cai, L. Single-cell systems biology by    super-resolution imaging and combinatorial labeling. Nat Methods 9,    743-8 (2012).-   15. Wei, B., Dai, M. & Yin, P. Complex shapes self-assembled from    single-stranded DNA tiles. Nature 485, 623-6 (2012).-   16. Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen    scavenging system for improvement of dye stability in    single-molecule fluorescence experiments. Biophys J 94, 1826-35    (2008).-   17. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and    long-lasting single-molecule fluorescence imaging. Nat Methods 3,    891-3 (2006).-   18. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional    super-resolution imaging by stochastic optical reconstruction    microscopy. Science 319, 810-3 (2008).-   19. Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green    fluorescent protein. Science 333, 642-6 (2011).-   20. Hein, B., Willig, K. I. & Hell, S. W. Stimulated emission    depletion (STED) nanoscopy of a fluorescent protein-labeled    organelle inside a living cell. Proc Natl Acad Sci USA 105, 14271-6    (2008).-   21. Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast,    three-dimensional super-resolution imaging of live cells. Nature    methods 8, 499-508 (2011).-   22. Willig, K. I. et al. Nanoscale resolution in GFP-based    microscopy. Nat Methods 3, 721-3 (2006).-   23. Stoltenburg, R., Reinemann, C. & Strehlitz, B. SELEX—a    (r)evolutionary method to generate high-affinity nucleic acid    ligands. Biomol Eng 24, 381-403 (2007).-   24. Paige, J. S., Nguyen-Duc, T., Song, W. & Jaffrey, S. R.    Fluorescence imaging of cellular metabolites with RNA. Science 335,    1194 (2012).-   25. Jaffrey, S. R. Personal Communication. Personal Communication    (2013).-   26. Fukusaki, E. et al. SELEX for tubulin affords specific T-rich    DNA aptamers. Systematic evolution of ligands by exponeential    enrichment. Bioorg Med Chem Lett 11, 2927-30 (2001).

What is claimed is:
 1. A protein-nucleic acid conjugate, comprising aprotein linked to a docking strand that is capable of transientlybinding to a complementary labeled imager strand.
 2. The protein-nucleicacid conjugate of claim 1, wherein the docking strand is transientlybound to the complementary labeled imager strand.
 3. The protein-nucleicacid conjugate of claim 1 or 2, wherein the protein is an antibody, anantigen-binding antibody fragment, or a peptide aptamer.
 4. Theprotein-nucleic acid conjugate of any one of claims 1-3, wherein theprotein is linked to the docking strand through an intermediate linker.5. The protein-nucleic acid conjugate of claim 4, wherein theintermediate linker comprises biotin and streptavidin.
 6. Theprotein-nucleic acid conjugate of any one of claims 3-5, wherein theantibody is a monoclonal antibody.
 7. The protein-nucleic acid conjugateof any one of claims 1-6, wherein the complementary labeled imagerstrand is a complementary fluorescently-labeled imager strand.
 8. Theprotein-nucleic acid conjugate of claim 7, wherein the complementaryfluorescently-labeled imager strand comprises at least one fluorophore.9. The protein-nucleic acid conjugate of any one of claims 1-8, whereinthe complementary labeled imager strand is about 4 to about 30nucleotides in length.
 10. The protein-nucleic acid conjugate of claim9, wherein the complementary labeled imager strand is about 8 to about10 nucleotides in length.
 11. A target bound to at least oneprotein-nucleic acid conjugate of any one of claims 1-10.
 12. The targetof claim 11, wherein the target is a protein.
 13. A plurality of theprotein-nucleic acid conjugates of any one of claims 1-10.
 14. Theplurality of claim 13, wherein the plurality comprises at least twosubsets of the protein-nucleic acid conjugates, and the protein-nucleicacid conjugates of each subset bind to different targets.
 15. Acomposition comprising the plurality of protein-nucleic acid conjugatesof claim 13 or 14, wherein at least one of the protein-nucleic acidconjugates is bound to at least one target.
 16. A compositioncomprising: at least one protein-nucleic acid conjugate that comprises aprotein linked to a docking strand, wherein the at least oneprotein-nucleic acid conjugate is bound to a target; and at least onecomplementary labeled imager strand that is transiently bound to the atleast one protein-nucleic acid conjugate.
 17. The composition of claim16, comprising at least two complementary labeled imager strands,wherein the at least two complementary labeled imager strands areidentical.
 18. The composition of claim 16, comprising at least twocomplementary labeled imager strands, wherein the at least twocomplementary labeled imager strands are different.
 19. The compositionof any one of claims 16-18, wherein the number of complementary labeledimager strands is less than, greater than or equal to the number ofprotein-nucleic acid conjugates.
 20. The composition of any one ofclaims 13-19, wherein the composition comprises at least 2 differentcomplementary labeled imager strands.
 21. The composition of any one ofclaims 13-20, wherein the composition comprises at least 5 differentcomplementary labeled imager strands.
 22. The composition of any one ofclaims 13-21, wherein the composition comprises at least 10 differentcomplementary labeled imager strands.
 23. The composition of claim 22,wherein the composition comprises at least 100 different complementarylabeled imager strands.
 24. The composition of any one of claims 16-23,wherein the complementary labeled imager strands are complementaryfluorescently-labeled imager strands.
 25. An antibody-DNA conjugate,comprising a monoclonal antibody linked to a docking strand that isbound to a complementary labeled imager strand, wherein the antibody andthe docking strand are each biotinylated and linked to each otherthrough a biotin-streptavidin linker.
 26. The antibody-DNA conjugate ofclaim 25, wherein the complementary labeled imager strand is acomplementary fluorescently-labeled imager strand.
 27. The antibody-DNAconjugate of any one of claims 1-26, wherein the docking strandcomprises at least two domains, wherein each domain binds to arespectively complementary labeled imager strand.
 28. The antibody-DNAconjugate of claim 27, wherein the docking strand comprises at leastthree domains, wherein each domain binds to a respectively complementarylabeled imager strand.
 29. The antibody-DNA conjugate of any one ofclaims 21-28, wherein the thermal stability of a docking strandtransiently bound to a complementary labeled imager strand is within 0.5kcal/mol of the thermal stability of other docking strands transientlybound to their respective labeled imager strands.
 30. An aptamer-nucleicacid conjugate, comprising a nucleic acid aptamer linked to a dockingstrand that is transiently bound to a complementary labeled imagerstrand.
 31. The aptamer-nucleic acid conjugate of claim 30, wherein thecomplementary labeled imager strand is a complementaryfluorescently-labeled imager strand.
 32. The aptamer-nucleic acidconjugate of claim 30 or 31, wherein the docking strand comprises atleast two domains, wherein each domain binds to a respectivelycomplementary labeled imager strand.
 33. The aptamer-nucleic acidconjugate of claim 32, wherein the docking strand comprises at leastthree domains, wherein each domain binds to a respectively complementarylabeled imager strand.
 34. The aptamer-nucleic acid conjugate of any oneof claims 30-33, wherein the thermal stability of a docking strandtransiently bound to a complementary labeled imager strand is within 0.5kcal/mol of the thermal stability of other docking strands transientlybound to their respective labeled imager strands.
 35. A method ofdetecting a target in a sample, the method comprising: contacting asample with (a) at least one protein-nucleic acid conjugate thatcomprises a protein linked to a docking strand and (b) at least onefluorescently-labeled imager strand that is complementary to andtransiently binds to the docking strand of the at least oneprotein-nucleic acid conjugate; and determining whether the at least oneprotein-nucleic acid conjugate binds to the target in the sample. 36.The method of claim 35, wherein the determining step comprises imagingtransient binding of the at least one fluorescently-labeled imagerstrand to the docking strand of the at least one protein-nucleic acidconjugate.
 37. The method of claim 35 or 36, wherein the protein of theprotein-nucleic acid conjugate is an antibody, an antigen-bindingantibody fragment, or a peptide aptamer.
 38. The method of claim 36,wherein the antibody is a monoclonal antibody.
 39. The method of any oneof claims 35-38, wherein the protein of the protein-nucleic acidconjugate is linked to the docking strand through an intermediatelinker.
 40. The method of claim 39, wherein the intermediate linkercomprises biotin and/or streptavidin.
 41. The method of any one ofclaims 35-40, wherein the complementary fluorescently-labeled imagerstrand comprises at least one fluorophore.
 42. The method of any one ofclaims 35-41, wherein the complementary fluorescently-labeled imagerstrand is about 4 to about 30 nucleotides in length.
 43. The method ofclaim 42, wherein the complementary fluorescently-labeled imager strandis about 8 to about 10 nucleotides in length.
 44. The method of any oneof claims 35-43, wherein the sample is a cell or cell lysate.
 45. Themethod of any one of claims 35-44, wherein the target is a protein 46.The method of any one of claims 35-45, wherein the target is obtainedfrom a cell or cell lysate.
 47. The method of any one of claims 35-46,wherein the docking strand comprises at least two domains, wherein eachdomain binds to a respectively complementary labeled imager strand. 48.The method of claim 47, wherein the docking strand comprises at leastthree domains, wherein each domain binds to a respectively complementarylabeled imager strand.
 49. The method of any one of claims 35-48,wherein the thermal stability of a docking strand transiently bound to alabeled imager strand is within 0.5 kcal/mol of the thermal stability ofother docking strands transiently bound to their respective labeledimager strands.
 50. A method of detecting at least one target in asample, the method comprising: contacting a sample with (a) at least twoprotein-nucleic acid conjugates, each comprising a protein linked to adocking strand, and (b) at least two labeled imager strands that arecomplementary to and transiently bind to respective docking strands ofthe at least two different protein-nucleic acid conjugates; anddetermining whether the at least two protein-nucleic acid conjugatesbind to at least one target in the sample.
 51. The method of claim 50,wherein the determining step comprises, in the following order, imagingtransient binding of one of the at least two labeled imager strands to adocking strand of one of the at least two protein-nucleic acidconjugates to produce a first image of signal, and imaging transientbinding of another of the at least two labeled imager strands to adocking strand of another of the at least two protein-nucleic acidconjugates to produce at least one other image of signal.
 52. The methodof claim 51, further comprising combining the first image and the atleast one other image to produce a composite image of signal, whereinthe signal of the composite image is representative of the at least onetarget.
 53. The method of any one of claims 50-52, wherein the proteinof the protein-nucleic acid conjugate is an antibody, an antigen-bindingantibody fragment, or a peptide aptamer.
 54. The method of claims 53,wherein the antibody is a monoclonal antibody.
 55. The method of any oneof claims 50-54 wherein the protein of the protein-nucleic acidconjugate is linked to the docking strand through an intermediatelinker.
 56. The method of claim 55, wherein the intermediate linkercomprises biotin and streptavidin.
 57. The method of any one of claims50-56, wherein each of the at least two labeled imager strands arefluorescently-labeled imager strands.
 58. The method of claim 57,wherein the at least two fluorescently-labeled imager strands arespectrally distinct, fluorescently-labeled imager strands.
 59. Themethod of any claim 57 or 58, wherein each of the at least two labeledimager strands comprises at least one fluorophore.
 60. The method of anyone of claims 50-59, wherein each of the at least two labeled imagerstrands is about 4 to about 30 nucleotides in length.
 61. The method ofclaim 60, wherein each of the at least two labeled imager strands isabout 8 to about 10 nucleotides in length.
 62. The method of any one ofclaims 50-61, wherein the sample is a cell or cell lysate.
 63. Themethod of any one of claims 50-62, wherein the at least one target is aprotein.
 64. The method of any one of claims 50-63, wherein the at leastone target is obtained from a cell or cell lysate.
 65. The method of anyone of claims 50-64, wherein step of determining whether the at leasttwo protein-nucleic acid conjugates bind to at least one target in thesample comprises determining whether the at least two protein-nucleicacid conjugates bind to at least two targets in the sample.
 66. Themethod of any one of claims 50-65, wherein the docking strand comprisesat least two domains, wherein each domain binds to a respectivelycomplementary labeled imager strand.
 67. The method of claim 66, whereinthe docking strand comprises at least three domains, wherein each domainbinds to a respectively complementary labeled imager strand.
 68. Themethod of any one of claims 50-67, wherein the thermal stability of adocking strand transiently bound to a labeled imager strand is within0.5 kcal/mol of the thermal stability of other docking strandstransiently bound to their respective labeled imager strands.
 69. Amethod of detecting at least one protein target in a sample, comprising:(a) contacting a sample with at least two protein-nucleic acidconjugates, each comprising a protein linked to a docking strand and (b)sequentially contacting the sample with at least two labeled imagerstrands that are complementary to and transiently bind to respectivedocking strands of the at least two protein-nucleic acid conjugates; anddetermining whether the at least two protein-nucleic acid conjugatesbind to at least one protein target in the sample.
 70. The method ofclaim 69, comprising, in the following ordered steps: contacting thesample with a first protein-nucleic acid conjugate and at least oneother protein-nucleic acid conjugate; contacting the sample with a firstlabeled imager strand that is complementary to and transiently binds tothe docking strand of the first protein-nucleic acid conjugate; imagingthe sample to obtain a first image, optionally using time-lapsedimaging; removing the first labeled imager strand; contacting the samplewith at least one other labeled imager strand that is complementary toand transiently binds to the docking strand of the at least one otherprotein-nucleic acid conjugate; and imaging the sample to obtain atleast one other image, optionally using time-lapsed imaging.
 71. Themethod of claim 69, comprising, in the following ordered steps:contacting the sample with a first protein-nucleic acid conjugate;contacting the sample with a first labeled imager strand that iscomplementary to and transiently binds to the docking strand of thefirst protein-nucleic acid conjugate; imaging the sample to obtain afirst image, optionally using time-lapsed imaging; removing the firstlabeled imager strand; contacting the sample with at least one otherprotein-nucleic acid conjugate; contacting the sample with at least oneother labeled imager strand that is complementary to and transientlybinds to the docking strand of the at least one other protein-nucleicacid conjugate; and imaging the sample to obtain at least one otherimage, optionally using time-lapsed imaging.
 72. The method of claim 70or 71, further comprising determining whether the first protein-DNAconjugate binds to a first target and/or whether the at least one otherprotein-DNA conjugate binds to at least one other target.
 73. The methodof claim 72, further comprising assigning a pseudo-color to the signalin the first image, and assigning at least one other pseudo-color to thesignal in the at least one other image.
 74. The method of claim 73,further comprising combining the first image and the at least one otherimage to produce a composite image of the pseudo-colored signals,wherein the pseudo-colored signals of the composite image arerepresentative of the at least two targets.
 75. The method of any one ofclaims 69-74, wherein the protein of the protein-nucleic acidconjugate(s) is an antibody, an antigen-binding antibody fragment, or apeptide aptamer.
 76. The method of claim 75, wherein the antibody is amonoclonal antibody.
 77. The method of any one of claims 69-76, whereinthe protein of the protein-nucleic acid conjugate(s) is linked to thedocking strand through an intermediate linker.
 78. The method of claim77, wherein the intermediate linker comprises biotin and/orstreptavidin.
 79. The method of any one of claims 69-78, wherein each ofthe labeled imager strands is a fluorescently-labeled imager strand. 80.The method of claim 69, wherein each of the labeled imager strands is aspectrally distinct, fluorescently-labeled imager strand.
 81. The methodof claim 69 or 70, wherein each of the labeled imager strands comprisesat least one fluorophore.
 82. The method of any one of claims 69-81,wherein each of the labeled imager strands is about 4 to about 30nucleotides in length.
 83. The method of claim 82, wherein each of thelabeled imager strands is about 8 to about 10 nucleotides in length. 84.The method of any one of claims 69-83, wherein the sample is a cell orcell lysate.
 85. The method of any one of claims 69-84, wherein thetarget(s) is a protein.
 86. The method of any one of claims 69-85,wherein the target(s) is obtained from a cell or cell lysate.
 87. Themethod of any one of claims 69-86, wherein the step of determiningwhether the at least two protein-nucleic acid conjugates bind to atleast one protein target in the sample comprises determining whether theat least two protein-nucleic acid conjugates bind to at least twoprotein targets in the sample.
 88. The method of any one of claims69-87, wherein each of the docking strand comprises at least twodomains, wherein each domain binds to a respectively complementarylabeled imager strand.
 89. The method of any one of claims 69-88,wherein each of the docking strand comprises at least three domains,wherein each domain binds to a respectively complementary labeled imagerstrand.
 90. The method of any one of claims 69-89, wherein the thermalstability of a docking strand transiently bound to a labeled imagerstrand is within 0.5 kcal/mol of the thermal stability of other dockingstrands transiently bound to their respective labeled imager strands.91. A method of detecting a molecule, comprising contacting a samplecontaining at least one target with (a) at least one BP-NA conjugate,each BP-NA conjugate comprising a binding partner linked to a dockingstrand and (b) at least one labeled imager strand that is complementaryto and transiently binds the docking strand of the at least one BP-NAconjugate; and determining whether the at least one BP-NA conjugatebinds to at least one target in the sample.
 92. The method of claim 91,wherein the determining step comprises imaging transient binding the atleast one labeled imager strand to the docking strand of the at leastone BP-NA conjugate.
 93. The method of claim 91 or 92, wherein thesample is a cell or cell lysate.
 94. The method of any one of claims91-93, wherein the at least one target is obtained from a cell or celllysate.
 95. The method of any one of claims 91-94, wherein the target isa naturally-occurring biomolecule.
 96. The method of claim 95, whereinthe naturally-occurring biomolecule is a protein.
 97. The method ofclaim 96, wherein the protein is an antibody, an antigen-bindingantibody fragment, or a peptide aptamer.
 98. The method of claim 97,wherein the antibody is a monoclonal antibody.
 99. The method of any oneof claims 91-98, wherein the target is linked to the docking strandthrough an intermediate linker.
 100. The method of claim 99, wherein theintermediate linker comprises biotin and/or streptavidin.
 101. Themethod of claim 95, wherein the naturally-occurring biomolecule is anucleic acid.
 102. The method of claim 101, wherein the nucleic acid isa nucleic acid aptamer.
 103. The method of any one of claims 91-103,wherein the labeled imager strand is a fluorescently-labeled imagerstrand.
 104. The method of claim 103, wherein the fluorescently-labeledimager strand comprises at least one fluorophore.
 105. The method of anyone of claims 91-104, wherein the fluorescently-labeled imager strand isabout 4 to about 30 nucleotides in length.
 106. The method of claim 105,wherein the fluorescently-labeled imager strand is about 8 to about 10nucleotides in length.
 107. The method of any one of claims 91-106,wherein the binding partner is a protein or nucleic acid.
 108. Themethod of any one of claims 91-107, wherein the docking strand comprisesat least two domains, wherein each domain binds to a respectivelycomplementary labeled imager strand.
 109. The method of claim 108,wherein the docking strand comprises at least three domains, whereineach domain binds to a respectively complementary labeled imager strand.110. The method of any one of claims 91-109, wherein the thermalstability of a docking strand transiently bound to a labeled imagerstrand is within 0.5 kcal/mol of the thermal stability of other dockingstrands transiently bound to their respective labeled imager strands.111. A method of detecting a naturally-occurring biomolecule, comprisingcontacting a sample containing at least one target with (a) at least twodifferent BP-NA conjugates, each BP-NA conjugate comprising a bindingpartner linked to a docking strand and (b) at least two labeled imagerstrands that are complementary to and transiently bind to respectivedocking strands of the at least two BP-NA conjugates; and determiningwhether the at least two BP-NA conjugates bind to at least one target inthe sample.
 112. The method of claim 111, comprising, in the followingordered steps: contacting the sample with a first BP-NA conjugate and atleast one other BP-NA conjugate; and contacting the sample with a firstlabeled imager strand that is complementary to and transiently binds tothe docking strand of the first BP-NA conjugate; imaging the sample toobtain a first image, optionally using time-lapsed imaging; removing thefirst fluorescently-labeled imager strand; contacting the sample with atleast one other labeled imager strand that is complementary to andtransiently binds to the docking strand of the at least one other BP-NAconjugate; and imaging the sample to obtain at least one other image,optionally using time-lapsed imaging.
 113. The method of claim 111,comprising, in the following ordered steps: contacting the sample with afirst BP-NA conjugate; contacting the sample with a first labeled imagerstrand that is complementary to and transiently binds to the dockingstrand of the first BP-NA conjugate; imaging the sample to obtain afirst image, optionally using time-lapsed imaging; removing the firstlabeled imager strand; contacting the sample with at least one otherBP-NA conjugate; contacting the sample with at least one other labeledimager strand that is complementary to and transiently binds to thedocking strand of the at least one other BP-NA conjugate; and imagingthe sample to obtain a at least one other image, optionally usingtime-lapsed imaging.
 114. The method of claim 112 or 113, furthercomprising determining whether the first protein DNA conjugate binds toa first target and/or whether the at least one other protein-DNAconjugate binds to at least one other target.
 115. The method of claim114, further comprising assigning a pseudo-color to the signal in thefirst image, and assigning at least one other pseudo-color to the signalin the at least one other image.
 116. The method of claim 115, furthercomprising combining the first image and the at least one other image toproduce a composite image of the pseudo-colored signals, wherein thepseudo-colored signals of the composite image are representative of theat least one target.
 117. The method of any one of claims 111-116,wherein the sample is a cell or cell lysate.
 118. The method of any oneof claims 111-117, wherein the at least one target is obtained from acell or cell lysate.
 119. The method of any one of claims 111-118,wherein the target is a naturally-occurring biomolecule.
 120. The methodof claim 119, wherein the naturally-occurring biomolecule is a protein.121. The method of claim 120, wherein the protein is an antibody, anantigen-binding antibody fragment, or a peptide aptamer.
 122. The methodof claim 121, wherein the antibody is a monoclonal antibody.
 123. Themethod of any one of claims 111-122, wherein the target is linked to thedocking strand through an intermediate linker.
 124. The method of claim123, wherein the intermediate linker comprises biotin and/orstreptavidin.
 125. The method of claim 119, wherein thenaturally-occurring biomolecule is a nucleic acid.
 126. The method ofclaim 125, wherein the nucleic acid is a nucleic acid aptamer.
 127. Themethod of any one of claims 111-126, wherein the first labeled imagerstrand is a fluorescently-labeled imager strand.
 128. The method of anyone of claims 111-126, wherein the at least one other labeled imagerstrand is a fluorescently-labeled imager strand.
 129. The method ofclaim 127 or 128, wherein the first labeled imager strand and/or the atleast one other labeled imager strand comprises at least onefluorophore.
 130. The method of any one of claims 111-129, wherein atleast one other labeled imager strand about 4 to about 30 nucleotides inlength.
 131. The method of claim 130, at least one other labeled imagerstrand is about 8 to about 10 nucleotides in length.
 132. The method ofany one of claims 111-131, wherein the binding partner is a protein ornucleic acid.
 133. The method of any one of claims 111-132, wherein thedocking strand is a DNA docking strand.
 134. The method of any one ofclaims 111-133, wherein the docking strand comprises at least twodomains, wherein each domain binds to a respectively complementarylabeled imager strand.
 135. The method of claim 134, wherein the dockingstrand comprises at least three domains, wherein each domain binds to arespectively complementary labeled imager strand.
 136. The method of anyone of claims 111-135, wherein the thermal stability of a docking strandtransiently bound to a labeled imager strand is within 0.5 kcal/mol ofthe thermal stability of other docking strands transiently bound totheir respective labeled imager strands.
 137. A method of determiningthe number of targets in a test sample, comprising: obtaining a samplethat comprises targets transiently bound directly or indirectly tolabeled imager strands; obtaining a time-lapsed image of the sample;performing spot detection and localization on the image to obtain ahigh-resolution image of the sample; calibrating k_(on)·c_(imager),wherein k_(on) is a second order association constant, and c_(imager) isconcentration of labeled imager strands in the test sample; determiningvariable τ_(d); and determining the number of test targets in the samplebased on the equation, number of testtargets=(k_(on)·C_(imager)·τ_(d))⁻¹.
 138. The method of claim 137,wherein the test targets are protein targets.
 139. The method of claim138, wherein the protein targets are bound to protein-nucleic acidconjugates that comprise a protein linked to a docking strand, and thelabeled imager strands are complementary to and transiently bind torespective docking strands of the protein-nucleic acid conjugates. 140.The method of claim 139, wherein the protein of the protein-nucleic acidconjugate is an antibody, an antigen-binding antibody fragment, or apeptide aptamer.
 141. The method of claim 137, wherein the test targetsare single-stranded nucleic acids.
 142. The method of claim 141, whereinthe single-stranded nucleic acids are DNA or RNA.
 143. The method of anyone of claims 137-142, wherein each of the labeled imager strands is afluorescently-labeled imager strand.
 144. The method of any one ofclaims 137-143, wherein each of the fluorescently-labeled imager strandscomprises at least one fluorophore.
 145. The method of any one of claims137-144, wherein each of the fluorescently-labeled imager strands isabout 3 to about 30 nucleotides in length.
 146. The method of claim 144,wherein each of the fluorescently-labeled imager strands is about 8 toabout 10 nucleotides in length.
 147. The method of any one of claims137-146, wherein the time-lapsed image is obtained over a period ofabout 24 minutes.
 148. The method of any one of claims 137-147, whereinthe time-lapsed image is a time-lapsed diffraction-limited fluorescenceimage.
 149. The method of any one of claims 137-148, wherein the step ofperforming localization on the image comprises performing Gaussianfitting on the image.
 150. The method of any one of claims 137-149,wherein the step of calibrating k_(on)·c_(imager) comprising calibratingk_(on)·c_(imager) with a control sample with a known number of targets.151. The method of any one of claims 137-150, wherein the step ofdetermining variable τ_(d) comprises determining variable τ_(d) byfitting the signal OFF-time distribution to a cumulative distributionfunction.
 152. The method of any one of claims 137-151, wherein thenumber of test targets is determined with an accuracy of greater than90%.
 153. A method of determining a relative amount of targets in a testsample, comprising: obtaining a sample that comprises targetstransiently bound directly or indirectly to labeled imager strands;obtaining a time-lapsed image of the sample; performing spot detectionand localization on the image to obtain a high-resolution image of thesample; determining variable τ_(d); and determining the relative amountof two or more test targets in the sample based on τ_(d).
 154. Themethod of claim 153, wherein the test targets are protein targets. 155.The method of claim 154, wherein the protein targets are bound toprotein-nucleic acid conjugates that comprise a protein linked to adocking strand, and the labeled imager strands are complementary to andtransiently bind to respective docking strands of the protein-nucleicacid conjugates.
 156. The method of claim 155, wherein the protein ofthe protein-nucleic acid conjugate is an antibody, an antigen-bindingantibody fragment, or a peptide aptamer.
 157. The method of claim 153,wherein the test targets are single-stranded nucleic acids.
 158. Themethod of claim 157, wherein the single-stranded nucleic acids are DNAor RNA.
 159. The method of any one of claims 153-158, wherein each ofthe labeled imager strands is a fluorescently-labeled imager strand.160. The method of claim 159, wherein wherein each of the labeled imagerstrands comprises at least one fluorophore.
 161. The method of any oneof claims 153-160, wherein each of the labeled imager strands is about 4to about 30 nucleotides in length.
 162. The method of claim 161, whereineach of the labeled imager strands is about 8 to about 10 nucleotides inlength.
 163. The method of any one of claims 153-162, wherein thetime-lapsed image is obtained over a period of about 25 minutes. 164.The method of any one of claims 153-163, wherein the time-lapsed imageis a time-lapsed diffraction-limited fluorescence image.
 165. The methodof any one of claims 153-164, wherein the step of performinglocalization on the image comprises performing Gaussian fitting on theimage.
 166. The method of any one of claims 153-165, wherein the step ofdetermining variable τ_(d) comprises determining variable τ_(d) byfitting the signal OFF-time distribution to a cumulative distributionfunction.
 167. The method of any one of claims 153-166, wherein thenumber of test targets is determined with an accuracy of greater than90%.
 168. A single-stranded DNA probe comprising a target binding domainof about 20 nucleotides in length linked to a docking domain comprisingat least one subdomain complementary to at least one labeled imagerstrand of about 4 to 30 nucleotides in length, and wherein the targetbinding domain is bound to a complementary domain of a single-strandedmRNA target strand.
 169. The single-stranded DNA probe of claim 168,wherein the at least one subdomain is transiently bound to the at leastone labeled imager strand.
 170. The single-stranded DNA probe of claim169, wherein the at least one labeled imager strand is fluorescentlylabeled.
 171. The single-stranded DNA probe of claim 170, wherein the atleast one labeled imager strand comprises a fluorophore.
 172. Thesingle-stranded DNA probe of claim 168, wherein the docking domaincomprises at least two subdomains, wherein the at least two subdomainsare respectively complementary to at least two labeled imager strands ofabout 4 to 30 nucleotides in length.
 173. The single-stranded DNA probeof claim 172, wherein the at least two subdomains are respectivelycomplementary to at least two labeled imager strands of about 8 to 10nucleotides in length.
 174. The single-stranded DNA probe of claim 172or 173, wherein the at least two subdomains are transiently bound torespectively complementary labeled imager strands.
 175. Thesingle-stranded DNA probe of any one of claims 172-174, wherein therespectively complementary labeled imager strands are distinctly labeledimager strands.
 176. The single-stranded DNA probe of any one of claims172-175, wherein the respectively complementary labeled imager strandsare respectively complementary fluorescently-labeled imager strands.177. The single-stranded DNA probe of claim 176, the respectivelycomplementary labeled imager strands each comprise a fluorophore. 178.The single-stranded DNA probe of claim 168, wherein the docking domaincomprises at least three subdomains, wherein the at least threesubdomains are respectively complementary to at least three labeledimager strands of about 4 to 30 nucleotides in length.
 179. Thesingle-stranded DNA probe of claim 178, wherein the at least threesubdomains are transiently bound to respectively complementary labeledimager strands.
 180. The single-stranded DNA probe of claim 179, whereinthe respectively complementary labeled imager strands are distinctlylabeled imager strands.
 181. The single-stranded DNA probe of claim 179or 180, wherein the respectively complementary labeled imager strandsare respectively complementary fluorescently-labeled imager strands.182. The single-stranded DNA probe of claim 180 or 181, wherein therespectively complementary labeled imager strands each comprise afluorophore.
 183. The single-stranded DNA probe of any one of claims168-182, wherein the target binding domain of about 20 nucleotides inlength is linked at its 3′ end to a docking domain.
 184. The Thesingle-stranded DNA probe of any one of claims 168-182, wherein thetarget binding domain of about 20 nucleotides in length is linked at its5′ end to a docking domain.
 185. A method of performing drift correctionfor a plurality of images, wherein each of the plurality of imagescomprises a frame of a time sequence of images, wherein the timesequence of images captures a plurality of transient events, the methodcomprising: determining a time trace for each of a plurality of driftmarkers identified in the plurality of images, wherein a time trace foreach drift marker corresponds to movement of an object in the image overthe time sequence of images; determining, with at least one computerprocessor, a first drift correction from at least one of the pluralityof drift markers based, at least in part, on the time traces for the atleast one of the plurality of drift markers; determining a time tracefor each of a plurality of geometrically-addressable marker sites from aplurality of drift templates identified from the plurality of images,wherein each drift template in the plurality of drift templatesdescribes a geometrical relationship between the plurality ofgeometrically-addressable marker sites of transient events in the drifttemplate; determining a second drift correction based, at least in part,on the time traces for the plurality of geometrically-addressable markersites from the plurality of drift templates; correcting the plurality ofimages based, at least in part, on the first drift correction and thesecond drift correction; and outputting a final image based on thecorrected plurality of images.
 186. The method of claim 185, furthercomprising: identifying a plurality of localizations in each of theplurality of images; creating a two-dimensional histogram of theplurality of localizations; and identifying locations of the pluralityof drift markers based, at least in part, on the two-dimensionalhistogram; wherein determining the time traces for each of the pluralityof drift markers comprises determining the time traces based, at leastin part, on the locations of the plurality of drift markers.
 187. Themethod of claim 186, wherein identifying a plurality of localizationscomprises: identifying a plurality of spots on each of the plurality ofimages; and determining a fitted center position of each of theplurality of spots using a local Gaussian fitting algorithm; whereineach of the plurality of localizations comprises the spot identified onan image and its associated fitted center position.
 188. The method ofclaim 187, wherein each of the plurality of localizations furthercomprises a detected photon count corresponding to the localization.189. The method of claim 186, wherein creating the two-dimensionalhistogram of the plurality of localizations comprises binning alllocalizations into a two-dimensional grid and using a total number oflocalizations in each bin as a histogram count.
 190. The method of claim186, wherein creating the two-dimensional histogram of the plurality oflocalizations comprises binning all localizations into a two-dimensionalgrid and using a total number of photon count of the plurality oflocalizations in each bin as a histogram count.
 191. The method of claim186, wherein identifying locations of the plurality of drift markersbased, at least in part, on the two-dimensional histogram comprises atleast one of the following: binarizing the two-dimensional histogramusing one or more selection criteria, wherein the one or more selectioncriteria include a lower-bound threshold of a histogram value or aupper-bound threshold of a histogram value; partitioning the binarizedimage into partitions and filtering the partitions based on one or moreselection criteria, wherein the one or more selection criteria includeone or more of a lower-bound threshold of an area of a partition area,an upper-bound threshold of the area, a lower-bound or an upper-bound ofa longest or shortest linear dimension of a partition longest, and alower-bound or an upper-bound of an eccentricity of a partition; andexpanding and shrinking the binarized image using one or more binaryimage operations, wherein the one or more binary image operationsinclude one or more of the following: dilate, erode, bridge, close,open, fill, clean, top-hat, bottom-hat, thicken, thin, and more. 192.The method of claim 185, wherein determining a first drift correctionbased, at least in part, on the time traces for the plurality of driftmarkers comprises: determining a relative time trace for each of theplurality of drift markers, wherein the relative time trace isdetermined by comparing the time trace for the drift marker with theaverage position of the same trace; and determining a combined timetrace based on the relative time traces for each of the plurality ofdrift markers; wherein determining the first drift correction based, atleast in part, on the time traces for the plurality of drift markerscomprises determining the first drift correction based, at least inpart, on the relative time traces for each of the plurality of driftmarkers.
 193. The method of claim 192, wherein determining the firstdrift correction based, at least in part, on the relative time tracesfor each of the plurality of drift markers comprises performing aweighted average of the relative time traces for each of the pluralityof drift markers.
 194. The method of claim 193, wherein performing theweighted average comprises: determining a quality score for each of therelative time traces, wherein the quality score is determined based, atleast in part, on a measure of variability over time associated with thetime trace and/or a measure of localization uncertainty of individuallocalizations within the time trace.
 195. The method of claim 194,wherein the measure of variability over time comprises a standarddeviation of the time trace over time.
 196. The method of claim 194,wherein the measure of localization uncertainty of individuallocalizations comprises, at least in part, an estimate of uncertaintyfrom a Gaussian fitting or a comparison with other simultaneouslocalizations, wherein the other simultaneous localizations are fromwithin a same image and from other time traces from the plurality ofdrift markers, wherein the comparison comprises a mean and standarddeviation of all simultaneous localizations.
 197. The method of claim185, further comprising: determining that a first drift marker of theplurality of drift markers is not present in at least one frame of thetime sequence of images; and linearly interpolating the time trace forthe first drift marker for the at least one frame to produce a smoothedtime trace for the first drift marker.
 198. The method of claim 185,wherein determining a time trace for each of a plurality ofgeometrically-addressable marker sites from a plurality of drifttemplates identified from the plurality of images comprises: identifyinga plurality of localizations in each of the plurality of images;creating a two-dimensional histogram of the plurality of localizations;and identifying the plurality of drift templates based, at least inpart, on the two-dimensional histogram; wherein identifying theplurality of drift templates comprises evaluating the two-dimensionalhistogram using an lower-bound and/or an upper-bound threshold in ahistogram count.
 199. The method of claim 185, wherein determining atime trace for each of a plurality of geometrically-addressable markersites from a plurality of drift templates identified from the pluralityof images comprises determining a time trace for each of a plurality ofgeometrically-addressable marker sites within each of the plurality ofdrift templates, and wherein determining the second drift correctioncomprises determining the second drift correction based, at least inpart, on the time traces for each of the plurality of marker siteswithin each of the plurality of drift templates.
 200. The method ofclaim 185, wherein determining the second drift correction based, atleast in part, on the time traces for each of the plurality ofgeometrically-addressable marker sites from each of the plurality ofdrift templates comprises: identifying a plurality ofgeometrically-addressable marker sites within each of the plurality ofdrift templates; and determining a relative time trace for each of aplurality of geometrically-addressable drift markers for each of theplurality of drift templates; wherein determining the second driftcorrection based, at least in part, on the time traces for the pluralityof drift templates comprises determining the second drift correctionbased, at least in part, on the relative time traces for each of theplurality of drift markers within each of the plurality of drifttemplates.
 201. The method of claim 200, wherein identifying a pluralityof geometrically addressable marker sites from each of the plurality ofdrift templates comprises determining a plurality of marker sites basedon, at least in part, a two-dimensional histogram of the plurality oflocalizations in the corresponding drift template, and/or one or moreselection criteria, wherein the one or more selection criteria includeone or more of a total number of localizations, a surface density oflocalizations, and standard deviation of localizations.
 202. The methodof claim 200, wherein determining the second drift correction based, atleast in part, on the relative time traces for each of the plurality ofdrift markers within each of the plurality of drift templates comprisesperforming a weighted average of the relative time traces for each ofthe plurality of drift markers within each of the drift templates. 203.The method of claim 202, wherein performing the weighted averagecomprises: determining a quality score for each of the relative timetraces, wherein the quality score is determined based, at least in part,on a measure of variability over time associated with the time traceand/or a localization uncertainty within the time trace.
 204. The methodof claim 203, wherein the measure of variability over time comprises astandard deviation of the time trace over time.
 205. The method of claim203, wherein the measure of localization uncertainty of individuallocalizations comprises an estimate of uncertainty from a Gaussianfitting or a comparison with other simultaneous localizations, whereinthe other simultaneous localizations are from within a same image andfrom the other time traces from the plurality of marker sites from theplurality of drift templates, wherein the comparison comprises a meanand standard deviation of all simultaneous localizations.
 206. Themethod of claim 185, wherein correcting the plurality of images based,at least in part, on the first drift correction and the second driftcorrection comprises correcting the plurality images using the firstdrift correction to produce a first corrected plurality of images, andwherein determining a time trace for each of a plurality of drifttemplates identified from the plurality of images comprises determininga time trace for each of the plurality of drift templates identifiedfrom the first corrected plurality of images.
 207. The method of claim185, further comprising: smoothing the first drift correction prior tocorrecting the plurality of images using the first drift correction.208. The method of claim 207, wherein smoothing the first driftcorrection comprises processing the first drift correction using localregression with a window determined by a characteristic drift time scaleof the first drift correction.
 209. The method of claim 185, furthercomprising smoothing the second drift correction prior to correcting theplurality of images using the second drift correction.
 210. The methodof claim 209, wherein smoothing the second drift correction comprisesprocessing the second drift correction using local regression with awindow determined by a characteristic drift time scale of the seconddrift correction.
 211. The method of claim 185, further comprising:selecting a single drift marker of the plurality of drift markers; anddetermining a third drift correction based, at least in part, on theselected single drift marker; wherein correcting the plurality of imagescomprises correcting the plurality of images based, at least in part, onthe third drift correction.
 212. The method of claim 211, whereincorrecting the plurality of images based, at least in part, on the thirddrift correction is performed prior to correcting the plurality ofimages based, at least in part on the first drift correction and thesecond drift correction.
 213. The method of any of claims 185 and 211,further comprising: identifying locations of a first plurality of pointsin a first image of the plurality of frames; identifying locations of asecond plurality of points in a second image of the plurality of images,wherein the second image corresponds to a neighboring frame of the firstimage in the time sequence of images; and determining a fourth driftcorrection based, at least in part, on differences between the locationsof the first plurality of points and the second plurality of points;wherein correcting the plurality of images comprises correcting theplurality of images based, at least in part, on the fourth driftcorrection.
 214. The method of claim 213, wherein the second imagecorresponds to a frame immediately following the frame corresponding tothe first image in the time sequence of images.
 215. The method of claim213, wherein determining the fourth drift correction based, at least inpart, on differences between the locations of the first plurality ofpoints and the second plurality of points comprises: creating ahistogram of distances between the locations of the first plurality ofpoints and the second plurality of points; determining based, at leastin part, on the histogram, pairs of points between the first image andthe second image that correspond to the same transient event; anddetermining a location offset between each of the determined pairs ofpoints; wherein determining the fourth drift correction is based on avector average of the location offsets for each of the determined pairsof points.
 216. The method of claim 185, wherein the plurality of imagescorrespond to DNA-based images and wherein the plurality of transientevents are binding events between an imaging strand and a DNA dockingstrand.
 217. The method of claim 216, wherein the imaging strand is afluorescent imaging probe configured to fluoresce when associated withthe DNA docking strand.
 218. The method of claim 185, wherein at leastone of the drift markers is a DNA based nanostructure.
 219. The methodof claim 218, wherein the DNA based nanostructure is a DNA origaminanostructure with docking strands.
 220. The method of claim 185,wherein at least one of the drift templates is a DNA basednanostructure.
 221. The method of claim 220, wherein the DNA basednanostructure is a DNA origami nanostructure with docking strands. 222.The method of claim 185, wherein at least one of the drift templates isa three-dimensional drift template.
 223. The method of claim 222,wherein the three-dimensional drift template is a tetrahedron.
 224. Themethod of claim 185, wherein at least one of the drift templatesincludes multiple colors corresponding to different types of transientevents.
 225. The method of claim 224, wherein the different types oftransient events include a first binding event of a first imaging strandwith a first type of DNA docking strand and a second binding event of asecond imaging strand with a second type of DNA docking strand.
 226. Themethod of claim 185, wherein outputting the final image comprisesdisplaying the final image on a display.
 227. The method of claim 185,wherein outputting the final image comprises sending the final image toa computer via at least one network.
 228. The method of claim 185,wherein outputting the final image comprises storing the final image onat least one storage device.
 229. A non-transitory computer readablemedium encoded with a plurality of instructions that, when executed byat least one computer processor, performs a method of performing driftcorrection for a plurality of images, wherein each of the plurality ofimages comprises a frame of a time sequence of images, wherein the timesequence of images captures a plurality of transient events, the methodcomprising: determining a time trace for each of a plurality of driftmarkers identified in the plurality of images, wherein a time trace foreach drift marker corresponds to movement of an object in the image overthe time sequence of images; determining a first drift correction fromat least one of the plurality of drift markers based, at least in part,on the time traces for the at least one of the plurality of driftmarkers; determining a time trace for each of a plurality ofgeometrically-addressable marker sites from a plurality of drifttemplates identified from the plurality of images, wherein each drifttemplate in the plurality of drift templates describes a geometricalrelationship between the plurality of geometrically-addressable markersites of transient events in the drift template; determining a seconddrift correction based, at least in part, on the time traces for theplurality of geometrically-addressable marker sites from the pluralityof drift templates; correcting the plurality of images based, at leastin part, on the first drift correction and the second drift correction;and outputting a final image based on the corrected plurality of images.230. A computer, comprising: an input interface configured to receive aplurality of images, wherein each of the plurality of images comprises aframe of a time sequence of images, wherein the time sequence of imagescaptures a plurality of transient events; at least one processorprogrammed to: determine a time trace for each of a plurality of driftmarkers identified in the plurality of images, wherein a time trace foreach drift marker corresponds to movement of an object in the image overthe time sequence of images; determine a first drift correction from atleast one of the plurality of drift markers based, at least in part, onthe time traces for the at least one of the plurality of drift markers;determine a time trace for each of a plurality ofgeometrically-addressable marker sites from a plurality of drifttemplates identified from the plurality of images, wherein each drifttemplate in the plurality of drift templates describes a geometricalrelationship between the plurality of geometrically-addressable markersites of transient events in the drift template; determine a seconddrift correction based, at least in part, on the time traces for theplurality of geometrically-addressable marker sites from the pluralityof drift templates; correct the plurality of images based, at least inpart, on the first drift correction and the second drift correction; anddetermine a final image based on the corrected plurality of images; andan output interface configured to output the final image.